Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

The purpose of this work is to address the relationship between transcriptional profile of embryos and the pregnancy success based on gene expression analysis of blastocyst biopsies taken prior to transfer to recipients. Biopsies (30-40% of the intact embryo) were taken from in vitro-produced day 7 blastocysts (n = 118), and 60-70% were transferred to recipients after reexpansion. Based on the success of pregnancy, biopsies were pooled in three groups (each 10 biopsies) namely: those resulting in no pregnancy (G1), resorbed embryos (G2), and those resulting in calf delivery (G3). Gene expression analysis of these groups was performed using home-made bovine preimplantation-specific cDNA array (219 clones) and BlueChip (with approximately 2,000 clones). Microarray data analysis results revealed a total of 52 and 58 genes were differentially regulated during comparison between G1 vs. G3 and G2 vs. G3. Biopsies resulted in calf delivery were enriched with genes necessary for implantation (COX2 and CDX2), carbohydrate metabolism (ALOX15), growth factor (BMP15), signal transduction (PLAU), and placenta-specific 8 (PLAC8). Biopsies from embryos resulting in resorption are enriched with transcripts involved protein phosphorylation (KRT8), plasma membrane (OCLN), and glucose metabolism (PGK1 and AKR1B1). Biopsies from embryos resulting in no pregnancy are enriched with transcripts involved inflammatory cytokines (TNF), protein amino acid binding (EEF1A1), transcription factors (MSX1, PTTG1), glucose metabolism (PGK1, AKR1B1), and CD9, which is an inhibitor of implantation. In conclusion, we generated direct candidates of blastocyst-specific genes which may play an important role in determining the fate of the embryo after transfer.

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.

Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30-40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

The accumulation of maternal mRNA and protein during oogenesis for supporting oocyte maturation and the newly fertilised zygote marks the beginning of developmental process in mammals. MicroRNAs (approximately 18-22 nt long) which are known for post-transcriptional gene regulation are evidenced for their essential role during animal development. We, therefore, aimed to investigate the expression of miRNAs in immature and in vitro matured bovine oocytes, using heterologous miRNA array platform. To attain this, we used a mercury locked nucleic acids (LNA) array (Exiqon, Vedbaek, Denmark) microarray that consist of 454 capture probes for human, mouse and rat miRNAs as registered and annotated in the miRBase release 8.0 at The Wellcome Trust Sanger Institute. Our result revealed the differential expression of 59 miRNAs, of which 31 and 28 miRNAs were found to be preferentially expressed in immature and matured oocytes, respectively. Here, we also report the identification of 32 orthologous miRNAs using a heterologous approach. Expression profiling of selected miRNAs during preimplantation stage embryos showed a distinct temporal expression pattern. After target prediction for selected candidate miRNAs high ranking target mRNA were quantified in immature and matured oocytes and showed a reciprocal expression pattern between the miRNA and the predicted targets suggesting a cause and effect relationship.