Project description:In vitro maturation (IVM) of the oocytes is a routine method in bovine embryo production. The competence of bovine oocytes to develop into embryo after IVM and in vitro fertilization (IVF) is lower as compared to in vivo preovulatory oocytes. Cumulus cells (CC) that enclose an oocyte are involved in the acquisition of oocyte quality during maturation. Using transcriptomic approach we compared cumulus cells gene expression during IVM with that in vivo preovulatory period.
Project description:In vitro maturation (IVM) of the oocytes is a routine method in bovine embryo production. The competence of bovine oocytes to develop into embryo after IVM and in vitro fertilization (IVF) is lower as compared to in vivo preovulatory oocytes. Cumulus cells (CC) that enclose an oocyte are involved in the acquisition of oocyte quality during maturation. Using transcriptomic approach we compared cumulus cells gene expression during IVM with that in vivo preovulatory period. Global transcriptional profiling was performed using cumulus cells collected from mature bovine oocytes (metaphase-II stage) after maturation performed either in vivo or in vitro. In vivo matured cumulus cells were collected from ovulatory follicles of Montbeliard adult cows by ovum pick-up in vivo (OPU, n=4). In vitro matured cumulus cells were recovered from the oocytes after 22h of in vitro culture of cumulus-oocyte complexes (50 COC per experiment) from 2-6 mm ovarian follicles of adult cows (MIV, n=4). Gene expression analysis was carried out between in vivo and in vitro matured cumulus representing a total of 8 slides (dye swap protocol)
Project description:Purpose: Preovualtory follicle diameter influences embryo quality in beef cattle that rely on a gonadotropin releasing hormone (GnRH) injection to induce the preovualtory gonadotropin surge and subsequent ovualtion. The goals of this study are to compare transcriptome profiles of pools of four oocytes or associated cumulus cells collected from small (≤11.7mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge and follicles (11.7-14.0 mm) exposed to an endogenous gonadotropin surge (spontaneous follicles). Methods: Oocyte and cumulus cell mRNA profiles of pools of four oocytes or associated cumulus cells collected from small (≤11.7mm) and large follicles (≥12.7 mm) exposed to a GnRH-induced gonadotropin surge or spontaneous follicles (11.7-14.0 mm) which were exposed to an endogenous gonadotropin surge were generated by deep sequencing (n=6 small follicle oocyte, 6 small follicle cumulus, 6 large follicle oocyte, 6 large follicle cumulus, 4 spontaneous follcile oocyte, and 5 spontaneous follicle cumulus pools), using Illumina HiSeq 2000 . The sequence reads that passed quality filters were aligned to the Bos taurus reference genome UMD3.1 using Hisat2mapper. FeatureCounts was used to quantify transcript abundance in each library using Bos taurus gene annotation from Ensembl. Differences in gene transcript expression were then analyzed amoung oocyte or cumulus cell follcile classifications using the packages edgeR and DESeq2 in R software. Results: We mapped an average of 30 million sequence reads per sample to the Bos taurus reference genome UMD3.1 and identified 69, 94, and 83 differentially expressed gene transcripts (DEG) among oocyte libraries from small versus large, small versus spontaneous, and large versus spontaneous follicle classifications, respectively. We also identified 128, 98, and 80 DEG among cumulus cell libraries from small versus large, small versus spontaneous, and large versus spontaneous follicles, respectively. Conclusions: Our study represents detailed analysis of RNA-sequencing data generated from in vivo produced cumulus and oocyte samples collected from preovualtory follicles of varied physiological status after exposure to an endogenous or exogenously stimulated gonadotropin surge. The results of our study highlight key differences in the transcriptome of samples from small, large, or spontaneous follicles and provide insight into the reduced embryo quality observed when small follicles undergo a GnRH induced gonadotropin surge.
Project description:During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the 4-cell, 8-cell, 16-cell and blastocyst stages. These were produced by in vitro fertilization of Bos t. taurus oocytes with sperm from a Bos t. indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 M-CM-^W 10^3 different genes were detected in the various developmental stages. EGA was analyzed by i) detection of embryonic transcripts which are not present in oocytes; ii) detection of transcripts from the paternal allele; and iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the 4-cell to the blastocyst stage. The largest proportion of gene activation, i.e. (i) 59%, (ii) 42%, and (iii) 58%, was found in 8-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the 4-cell stage identified categories related to translation, RNA processing and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and new insight into the timing of embryonic activation of specific genes. RNA-Seq profiles from pools of 10 ooytes/embryos from bovine Bos t. taurus GV and MII oocytes and a cross-breed of Bos t. taurus x Bos t. indicus from 4-cell, 8-cell, 16-cell and blastocysts generated using Illumina GAIIx
Project description:Study the effect of in vitro maturation system on the transcriptomics of bovine cumulus cells during incubation of first 6 hrs. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) after 6 hrs of in vitro matutaion(Ct-6H) vs. cumulus cells before maturation (Ct-0H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:The oocyte forms a complex with their somatic cumulus cells within the follicle throughout the preovulatory maturation steps. Cumulus cells support their oocyte not only through mechanical protection but also with a close bidirectional exchange of metabolites. Analysis of the oocytes cumulus gives the opportunity to explore non-invasively oocytal well-being and quality. In vitro maturation (IVM) is the first rate-limiting step in in vitro embryo production. Analysis of protein expression in cumulus cells around this critical step helps to explore the impact of maturation conditions and to examine an influence on maturational competence of the oocyte. The goal of this study was the comparison of the cumulus proteome of oocytes with and without maturational competence matured under in vivo and in vitro conditions. Therefore twenty cumulus samples corresponding to single oocytes were analysed. Half of the samples were matured in vivo and the other half in vitro. For each maturation group, cumulus from oocytes matured successfully (SM; n=5) and failed to mature (FM; n=5) were analysed.
Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.