Methods
Tissue expression profiles
All intensity values and their associated metadata were retrieved from the ELMA database.
All arrays/channels were assigned to a condition/group according to their metadata and manual curation.
Raw intensities of the (-)3xSLv1 negative controls were used to determine a per-array/channel background threshold,
defined as the mean of the negative control intensities + 2 times the standard deviation of those same intensities.
This background threshold was subtracted from all probe intensity values. The resulting background relative intensities
are plotted on the y axis.
All intensities were then quantile normalized using the
limma bioconductor package. Normalized intensity values were used to produce the color-scale used for points within the profiles.
For each probe, ad hoc statistical tests were performed comparing specific conditions within groups.
For groups with two conditions, a t-test was performed. For groups with 3 or more conditions a fixed-effect ANOVA was performed.
All p-values were then subjected to a unified FDR correction. Comparisons where the corrected p-value was below 0.05 are
highlighted in red in the resulting graphs.
Time course of early development
Library preparation
Three pools of 20 oocytes or 20 embryos were collected for each developmental stage. Total RNA extraction and DNAse I treatment were performed using PicoPure columns (Life Technologies). The synthetic transcriptome ERCC (Life Technologies) was spiked in the extraction buffer that was distributed equally in all samples. Next, cDNA first and second-strand synthesis was carried out using NuGen’s Ovation kit. The cDNA was then amplified with NuGen’s SPIA system. The final cDNA product was fragmented, ligated, and primed with bar-coded adaptors for RNAseq using the Encore kit (NuGen). Libraries were pooled and sequencing reactions were carried out on a HiSeq2000 system (Illumina) for 200 cycles (Genome Quebec Innovation Center). The exogenous RNA spike-in control mix was used for normalization to account for sample loss during sample processing and to recapitulate the natural difference in total RNA content found between developmental stages.
Bioinformatic analysis
Raw reads were processed to remove read-through Illumina primers and the low-quality ends of sequences using the Cutadapt software. Cleaned sequences with a length smaller than 30 bp were then removed using Sickle (https://github.com/ucdavis-bioinformatics/sickle/). Average read length after cleanup and removal of read-through adaptors was 77 bp. Sequences were aligned to the UMD3.1 assembly of the bovine genome using TopHat2, and transcript abundances were quantified against the reference transcriptome using Cufflinks2. Transcript abundances in FPKM were normalized against ERCC abundances in each library to obtain absolute estimates of the expression levels of each transcript which can be compared between developmental stages.
References
For more information about the EmbryoGENE transcriptomic platform, the EMBV3 array and its related protocols, refer to the following article: Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.
For more information about the individual experiments which form the basis for the experimental groups, consult the following articles published in peer-reviewed journals:
Blastocysts
Metabolism related
| Sample group | Reference | | Glucose (Blastocysts) | Cagnone G., Dufort I., Vigneault C., Sirard M., 2012. Differential gene expression profile in bovine blastocysts resulting from hyperglycemia exposure during early cleavage stages.Biology of reproduction 1(86) |
| Insulin | Laskowski D., Sjunnesson Y., Humblot P., Sirard M., Andersson G., Gustafsson H., Båge R., 2016. Insulin exposure during in vitro bovine oocyte maturation changes blastocyst gene expression and developmental potential.Reproduction, fertility, and development |
| Effects of Carnitine | Baldoceda L., Gagné D., Ferreira C., Robert C., 2015. Genetic influence on the reduction in bovine embryo lipid content by l-carnitine.Reproduction, fertility, and development |
| Effects of Vitamin K | Baldoceda-Baldeon L., Gagné D., Vigneault C., Blondin P., Robert C., 2014. Improvement of bovine in vitro embryo production by vitamin K₂ supplementation.Reproduction (Cambridge, England) 1(148) |
| Free radicals | Cagnone G., Sirard M., 2013. Transcriptomic signature to oxidative stress exposure at the time of embryonic genome activation in bovine blastocysts.Molecular reproduction and development 1(80) |
| Lipid effects (combined) | Van Hoeck V., Rizos D., Gutierrez-Adan A., Pintelon I., Jorssen E., Dufort I., Sirard M., Verlaet A., Hermans N., Bols P., Leroy J., 2015. Interaction between differential gene expression profile and phenotype in bovine blastocysts originating from oocytes exposed to elevated non-esterified fatty acid concentrations.Reproduction, fertility, and development 1(27) |
| Lipid effects (serum) | Cagnone G., Sirard M., 2014. The impact of exposure to serum lipids during in vitro culture on the transcriptome of bovine blastocysts.Theriogenology 1(81) |
Assisted Reproduction Techniques
| Sample group | Reference | | Effects of SAM | Responses of bovine early embryos to S-adenosyl methionine supplementation in culture (In press) |
| Vitrification (Bos indicus) | de Oliveira Leme L., Dufort I., Spricigo J., Braga T., Sirard M., Franco M., Dode M., 2016. Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos.Theriogenology 1(85) |
| Cloning | Hosseini S., Dufort I., Nieminen J., Moulavi F., Ghanaei H., Hajian M., Jafarpour F., Forouzanfar M., Gourbai H., Shahverdi A., Nasr-Esfahani M., Sirard M., 2016. Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.BMC genomics (17) |
| Culture conditions | Plourde D., Vigneault C., Lemay A., Breton L., Gagné D., Laflamme I., Blondin P., Robert C., 2012. Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts.Theriogenology 1(78) |
| Culture conditions | Plourde D., Vigneault C., Laflamme I., Blondin P., Robert C., 2012. Cellular and molecular characterization of the impact of laboratory setup on bovine in vitro embryo production.Theriogenology 1(77) |
| Effects of CSF2 | Dobbs K., Gagné D., Fournier E., Dufort I., Robert C., Block J., Sirard M., Bonilla L., Ealy A., Loureiro B., Hansen P., 2014. Sexual dimorphism in developmental programming of the bovine preimplantation embryo caused by colony-stimulating factor 2.Biology of reproduction 1(91) |
| Impact of culture | Unpublished |
Other
| Sample group | Reference | | Breed effect on IVC | Baldoceda L., Gilbert I., Gagné D., Vigneault C., Blondin P., Ferreira C., Robert C., 2015. Breed-specific factors influence embryonic lipid composition: comparison between Jersey and Holstein.Reproduction, fertility, and development |
| Effects of inbreeding | Unpublished |
| Donor age | Unpublished |
| ICM/Trophectoderm contrast | Hosseini S., Dufort I., Caballero J., Moulavi F., Ghanaei H., Sirard M., 2015. Transcriptome profiling of bovine inner cell mass and trophectoderm derived from in vivo generated blastocysts.BMC developmental biology (15) |
Granulosa
Follicles over time
| Sample group | Reference | 6-9mm Follicles
(follicular state effect) | Douville G., Sirard M., 2014. Changes in granulosa cells gene expression associated with growth, plateau and atretic phases in medium bovine follicles.Journal of ovarian research (7) |
>9mm Follicles
(follicular state effect) | Girard A., Dufort I., Douville G., Sirard M., 2015. Global gene expression in granulosa cells of growing, plateau and atretic dominant follicles in cattle.Reproductive biology and endocrinology : RB&E (13) |
In vitro culture
(FSH effect) | Unpublished |
Aromatase
expression level | A Genetical Genomics Methodology to Identify Genetic Markers of a Bovine Fertility Phenotype Based on CYP19A1 Gene Expression |
Dominant follicle
(age effect) | Unpublished |
Dominant follicle
(coasting effect) | Nivet A., Bunel A., Labrecque R., Belanger J., Vigneault C., Blondin P., Sirard M., 2012. FSH withdrawal improves developmental competence of oocytes in the bovine model.Reproduction (Cambridge, England) 1(143) |
Dominant follicle
(time to LH surge) | Gilbert I., Robert C., Vigneault C., Blondin P., Sirard M., 2012. Impact of the LH surge on granulosa cell transcript levels as markers of oocyte developmental competence in cattle.Reproduction (Cambridge, England) 1(143) |
Preovulatory follicle
(24h post-LH
age effect) | Dias F., Khan M., Adams G., Sirard M., Singh J., 2014. Granulosa cell function and oocyte competence: Super-follicles, super-moms and super-stimulation in cattle.Animal reproduction science 1(149) |
Superstimulated
preovulatory follicle
(24h post-LH) | Dias F., Khan M., Sirard M., Adams G., Singh J., 2013. Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle.Reproduction (Cambridge, England) 1(146) |
Dominant follicles (post-partum cows)
Oocyte
Oocytes
| Sample group | Reference | | Coasting time | Labrecque R., Vigneault C., Blondin P., Sirard M., 2014. Gene expression analysis of bovine oocytes at optimal coasting time combined with GnRH antagonist during the no-FSH period.Theriogenology 1(81) |
| Chromatin configuration | Labrecque R., Lodde V., Dieci C., Tessaro I., Luciano A., Sirard M., 2015. Chromatin remodelling and histone m RNA accumulation in bovine germinal vesicle oocytes.Molecular reproduction and development 1(82) |
| Follicle size | Labrecque R., Fournier E., Sirard M., 2016. Transcriptome analysis of bovine oocytes from distinct follicle sizes: Insights from correlation network analysis.Molecular reproduction and development 1(83) |
| PolyA tail length | Gohin M., Fournier E., Dufort I., Sirard M., 2014. Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes.Molecular human reproduction 1(20) |
Cumulus cells
| Sample group | Reference | | Coasting time | Gohin M., Fournier E., Dufort I., Sirard M., 2014. Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes.Molecular human reproduction 1(20) |
| Chromatin state | Dieci C., Lodde V., Labreque R., Dufort I., Tessaro I., Sirard M., Luciano A., 2016. Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.Molecular human reproduction 1(22) |
| Effects of cAMP | Khan D., Guillemette C., Sirard M., Richard F., 2015. Transcriptomic analysis of cyclic AMP response in bovine cumulus cells.Physiological genomics 1(47) |