Genes associated with survival of female bovine blastocysts produced in vivo.

The objective was to characterize the transcriptome profile of in vivo-derived female embryos competent to establish and maintain gestation. Blastocysts from superovulated heifers were bisected to generate two demi-embryos. One demi-embryo was transferred into a synchronized recipient and the other part was used for RNA-seq analysis. Data on transcript abundance was analyzed for 4 demi-embryos that established and maintained pregnancy to day 60 (designated as PP) and 3 that did not result in a pregnancy at day 30 (designated as NP). Using a false discovery rate of P < 0.10 as cutoff, a total of 155 genes were differentially expressed between PP and NP embryos, of which 73 genes were upregulated and 82 genes were downregulated in the PP group. The functional cluster with the greatest enrichment score for embryos that survived, representing 28 genes (48% of the annotated genes), was related to membrane proteins, particularly those related to olfaction and neural development and function. The functional cluster with the greatest enrichment score for downregulated genes in embryos that survived included terms related to oxidative phosphorylation, mitochondrial function, and transmembrane proteins. In conclusion, competence of in vivo-derived female bovine embryos to survive after transfer is associated with increased expression of genes encoding transmembrane proteins, perhaps indicative of differentiation of the inner cell mass to epiblast, and decreased expression of genes involved in oxidative phosphorylation, perhaps indicative of reduced metabolic activity.

Correction to: Genes associated with survival of female bovine blastocysts produced in vivo.

The first error is on page 5. A sentence lists two genes as SCNA1A and SCNA2A but they should be SCN1A and SCN2A.

Molecular signatures of bovine embryo developmental competence.

Assessment of the developmental capacity of early bovine embryos is still an obstacle. Therefore, the present paper reviews all current knowledge with respect to morphological criteria and environmental factors that affect embryo quality. The molecular signature of an oocyte or embryo is considered to reflect its quality and to predict its subsequent developmental capacity. Therefore, the primary aim of the present review is to provide an overview of reported correlations between molecular signatures and developmental competence. A secondary aim of this paper is to present some new strategies to enable concomitant evaluation of the molecular signatures of specific embryos and individual developmental capacity.

Expression analysis of regulatory microRNAs in bovine cumulus oocyte complex and preimplantation embryos.

MicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

Expression of microRNA and microRNA processing machinery genes during early quail (Coturnix japonica) embryo development.

MicroRNA (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNA and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6, and 9 of embryo development (19, 24, and 30 h of incubation, respectively). Total RNA including miRNA was isolated from 4 groups of embryos (each group consisting of 6 to 8 embryos) were collected at each of the 3 stages (19, 24, and 30 h). The expression pattern of candidate miRNA and miRNA processing machinery genes was performed using quantitative real-time PCR. The results demonstrated that 7 miRNA (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a, and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition, the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared with both 19 and 24 h. Our results suggest that machinery genes for miRNA biogenetic pathways are functional, and hence, miRNA may be involved in the regulation of early quail development. These 7 differentially expressed miRNA are suggested to play critical roles in quail embryo somite formation.

Effect of reproductive tract environment following controlled ovarian hyperstimulation treatment on embryo development and global transcriptome profile of blastocysts: implications for animal breeding and human assisted reproduction.

BACKGROUND: In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers. METHODS: Nineteen Simmental heifers were synchronized, superovulated and artificially inseminated; nine heifers were flushed on Day 2 after insemination and 2-4-cell stage embryos were recovered and endoscopicaly transferred to the ipsilateral oviduct of unstimulated (i.e. single-ovulating) synchronized recipients (n= 4 recipients; 25-50 embryos per recipient). The remaining 10 superovulated heifers and the unstimulated recipients were then non-surgically flushed on Day 7 to collect embryos. The blastocyst transcriptome profile was examined using the Affymetrix GeneChip Bovine Genome Array. RESULTS: The proportion of embryos, which developed to the blastocyst stage, was lower in superovulated heifers than unstimulated heifers (P< 0.05). Blastocysts that developed under the abnormal endocrine conditions associated with ovulation induction showed higher cellular and metabolic activities, as genes involved in the oxidative phosphorylation pathway, different metabolic processes and translation and transcription processes, in addition to genes expressed in response to stress, were highly expressed compared with embryos that developed in the oviduct of unstimulated animals. CONCLUSIONS: The environment in which the embryo develops in the oviduct/uterus significantly alters gene expression patterns, especially those genes that regulate metabolic activity in the embryo.

Bovine blastocysts with developmental competence to term share similar expression of developmentally important genes although derived from different culture environments.

This study was conducted to investigate the gene expression profile of in vivo-derived bovine embryo biopsies based on pregnancy outcomes after transferring to recipients. For this, biopsies of 30-40% embryos were taken from grade I blastocysts (International Embryo Transfer Society Manual) and the remaining 60-70% of the intact embryos were transferred to recipients. Frozen biopsies were pooled into three distinct groups based on the pregnancy outcome after transferring the corresponding parts, namely those resulting in no pregnancy (NP), pregnancy loss (PL), and calf delivery (CD). Array analysis revealed a total of 41 and 43 genes to be differentially expressed between biopsies derived from blastocysts resulting in NP versus CD and PL versus CD respectively. Genes regulating placental development and embryo maternal interaction (PLAC8) were found to be upregulated in embryo biopsies that ended up with CD. Embryo biopsies that failed to induce pregnancy were enriched with mitochondrial transcripts (Fl405) and stress-related genes (HSPD1). Overall, gene expression profiles of blastocysts resulting in NP and CD shared similar expression profiles with respect to genes playing significant roles in preimplantation development of embryo. Finally, comparing the transcript signatures of in vivo- and in vitro-derived embryos with developmental competence to term revealed a similarity in the relative abundance of 18 genes. Therefore, we were able to present a genetic signature associated with term developmental competence independent of the environmental origin of the transferred blastocysts.

Cryosurvival of in vitro produced bovine embryos supplemented with l-Carnitine and concurrent reduction of fatty acids.

Lipid accumulation is associated with reduced embryonic quality, causing limited survival after cryopreservation. Therefore, in the present study we aimed to reveal the effects of supplementation of a lipid reducing agent, l-carnitine and the removal of fatty acids during in vitro culture on the morphological as well as on the molecular level. To accomplish that, presumptive zygotes were cultured in 4 contrasting groups: namely SOFaa medium supplemented with BSA, (BSA), SOFaa medium supplemented with fatty acid free BSA (FAF), SOFaa medium supplemented with BSA as well as l-Carnitine (BSA + LC) and SOFaa medium concurrently supplemented with fatty acid free BSA and l-Carnitine (FAF + LC). Considering the developmental rates, no impact of different treatments was observed. Conversely, treatment groups clearly affected lipid content, with the lowest amounts detected in embryos derived from FAF and BSA + LC groups, implicating that both removal of fatty acids and supplementation of LC reduces lipid content effectively. Importantly, survival rates after cryopreservation show that LC significantly affects the kinetics of re-expansion, with the highest hatching rates detected for embryos cultured in FAF + LC (p < 0.05). Noteworthy, the highest cryotolerance did not go along with lowest lipid contents. Finally, metabolic alterations between the groups were reflected in different abundances of selected candidate genes related to lipid metabolism and oxidative stress response, like AMPKA1, ACC and PGC1 α or KEAP1 and SOD1. All in all, highly beneficial effects on survival rates after cryopreservation have been detected when embryos were cultured in absence of fatty acids and concurrent presence of l-Carnitine. Highest cryotolerance, however, did not correlate with lowest lipid contents.

Birth of healthy calves after intra-follicular transfer (IFOT) of slaughterhouse derived immature bovine oocytes.

To circumvent the negative impacts of in vitro culture on bovine embryos, we have recently established a new method, the so called intra-follicular oocyte transfer (IFOT), enabling in vivo fertilization and in vivo development of in vitro matured oocytes up to the blastocyst stage as well as to term. In this study, we raised the question whether immature bovine oocytes could also be transferred into a pre-ovulatory follicle to support in vivo maturation prior to subsequent in vivo fertilization, in vivo development as well as to term. To unravel that question, a total of 791 immature oocytes were transferred in groups of ∼50 into pre-ovulatory follicles of 16 recipient heifers. Consequently, we were able to recollect a total of 306 structures 8 days thereafter (38.5%). All in all, 12 heifers (75%) gave embryos developed to the morula or blastocyst stage in addition to the expected native embryos. Among all recollected structures, 40.1% had developed to the morula and/or blastocyst stage, meaning a total efficiency of 17.3% based on all transferred oocytes. Of impact, IFOT-embryos reached significantly higher developmental rates to the Morula and/or blastocyst stage until day 7 compared to in vitro cultured control embryos, despite being derived from the same charge of slaughterhouse ovaries (40.1 vs. 29.3%). This implicates a beneficial effect of the follicular environment for the intrinsic quality of the fertilized embryos during maturation and for subsequent developmental rates up to the blastocyst stage. Finally, the birth of two healthy calves after transfer of frozen-thawed IFOT-derived blastocysts to final recipients established the first proof of principle that IFOT of immature bovine oocytes generates bovine blastocysts bearing developmental capacity to term. Likewise, to the best of our knowledge, these calves are the first calves derived from full in vivo development of immature slaughterhouse derived oocytes. Thus, the results of the present study clearly demonstrate that IFOT of immature slaughterhouse-derived oocytes is now a feasible technique. Since efficiencies following IFOT achieved within the present study were improved compared to previous studies, IFOT now offers an attractive option for designing new scientific experiments.

The role of microRNAs in mammalian oocytes and embryos.

Advanced genomic analysis has revealed an enormous inventory of non-coding RNAs (ncRNAs), which are functionally important at transcriptional and post-transcriptional level for different cellular processes. Among the ncRNAs, microRNAs (miRNAs) have recently been highlighted extensively for their pivotal role in disease, fertility and development through post-transcriptional regulation of gene expression. The presence and spatio-temporal expression of miRNAs and miRNA processing machinery genes in oocytes and preimplantation embryos has evidenced the involvement of miRNAs for growth and maturation of mammalian oocytes, early embryonic development, stem cell lineage differentiation and implantation. Therefore, this article aims to highlight primary evidences on the importance of miRNAs and their mediated translational reprogramming in the physiology and development of mammalian oocytes and embryos.

Incidence of apoptosis and transcript abundance in bovine follicular cells is associated with the quality of the enclosed oocyte.

The close contact and interaction between the oocyte and the follicular environment influence the establishment of oocyte developmental competence. Moreover, it is assumed that apoptosis in the follicular cells has a beneficial influence on the developmental competence of oocytes. The aim of this study was to investigate whether bovine oocytes with varied developmental competence show differences in the degree of apoptosis and gene expression pattern in their surrounding follicular cells (cumulus and granulosa cells). Oocytes and follicular cells from follicles of 3 to 5 mm in diameter were grouped as brilliant cresyl blue (BCB)+ and BCB- based on glucose-6-phosphate dehydrogenase (G6PDH) activity in the ooplasm by BCB staining. In the follicular cells initial, early and late apoptotic events were assessed by analyzing caspase-3 activity, annexin-V and TUNEL, respectively. Global gene expression was investigated in immature oocytes and corresponding follicular cells. BCB+ oocytes resulted in a higher blastocyst rate (19.3%) compared to the BCB- group (7.4%, P < 0.05). Moreover, the analysis of apoptosis showed a higher caspase-3 activity in the follicular cells and an increased degree of late apoptotic events in granulosa cells in the BCB+ compared with the BCB- group. Additionally, the global gene expression profile revealed a total of 34 and 37 differentially expressed genes between BCB+ and BCB- cumulus cells and granulosa cells, respectively, whereas 207 genes showed an altered transcript abundance between BCB+ and BCB- oocytes. Among these, EIF3F, RARRES2, RNF34, ACTA1, GSTA1, EIF3A, VIM and CS gene transcripts were most highly enriched in the BCB+ oocytes, whereas OLFM1, LINGO1, ALDH1A3, PTHLH, BTN3A3, MRPS2 and PPM1K were most significantly reduced in these cells. Therefore, the follicular cells enclosing developmentally competent oocytes show a higher level of apoptosis and a different pattern of gene expression compared to follicular cells enclosing non-competent bovine oocytes.

G6PDH-activity in equine oocytes correlates with morphology, expression of candidate genes for viability, and preimplantative in vitro development.

Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) maturational competence; (3) gene expression of certain molecular markers; and (4) in vitro embryo development after intracytoplasmic sperm injection. Equine oocytes were exposed to BCB stain and were classified as BCB+ or BCB- according to their ability to convert the dye from blue to colorless. Additionally, BCB+ and BCB- oocytes were subclassified as having a compact (Cp) or expanded (Ex) cumulus complex. As a result, BCB+ oocytes had a greater proportion of expanded cumulus oocyte complexes compared with BCB- oocytes (71.2% vs. 49.5%). Moreover, we observed a significant difference in oocyte diameter and volume between BCB+ and BCB- oocytes irrespective of cumulus morphology. BCB+ oocytes reached a higher maturation rate compared with BCB- oocytes (59.0% vs. 28.7%). Regarding the analyzed candidate genes, relative transcript abundance was significantly different for nine genes. The expression of eight genes was significantly higher (P < 0.05) for BCB+ oocytes, including ATPV6E, IF-3, TFAM, DNMT1, STAT3, Aurora-A, ODC1, and CKS2 whereas BCB- oocytes showed higher in expression of COX1. These results are in line with the observed developmental competence. Cleavage rate (45.9% vs. 29.0%) and percentage of embryos that reached the blastocyst stage (9.2% vs. 1.4%) were significantly higher for embryos derived from BCB+ oocytes compared with BCB- oocytes. In conclusion, the present study provides evidence that G6PDH-activity in immature equine oocytes is a useful predictor for subsequent in vitro developmental competence.