Da Silveira, J. C. | bioRxiv Science

bioRxiv2023

Assessment of Total Oocyte Transcripts Representation through Single Ooplasm Biopsy in Bovine with High Reliability

Understanding the entire transcriptional and epigenetic landscape is facilitated by the application of omics in a number of ways. Today, omic instruments are more affordable and easier to implement. In human research, for instance, single-omics are a reality and are used extensively to generate vast quantities of data. This method permits the comprehensive reconstruction of transcriptome and epigenetic markers removing bias from pooled samples. In tandem with the evolution of machines and protocols, algorithms and genome annotation have undergone continuous improvement. The genome annotation of domestic animals is inferior to that of humans, rodents, and less complex organisms. In the case of heifers, the reference is incomplete, with significant gaps and only a portion of the noncoding transcripts. The purpose of this study is to validate our compartmentalized single oocyte biopsy by comparing a small fraction of bovine oocytes, 1%, to the entire oocyte at the Metaphase II stage. In addition, we examined the use of four database sources (NCBI, ENSEMBL, UCSC, and NONCODE) to produce a merged non-redundant gene alignment and counting in order to enhance gene detection and normalization, resulting in a more accurate method to comprehend the entire landscape. This study is a continuation of our research titled "Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine" in which this method was used to retrospectively compare biopsy oocytes collected during the MII phase. With the addition of NONCODE information, gene normalization was significantly enhanced. In addition, our analysis identified 4560 noncoding genes from NONCODE references. ENSEMBL and NCBI have nearly the same number of annotated genes (16,423 vs. 17,804), but using ENSEMBL as a reference, 2356 genes were able to be normalized and identified. Proceeding to biopsy x oocyte analysis, we were able to detect a greater number of genes in oocytes than in biopsy, where the preponderance was from NONCODE sources (68). Despite these minor differences, the high correlation of expression between them (89%) was consistent and proved to be a valuable instrument for studying the oocyte without destroying it.

bioinformatics↗

bioRxiv2023

Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine

Developmental competence is obtained by a series of morphological and molecular changes during mammalian oocyte growth within the ovulatory follicle. This entails the accumulation of cytoplasmic transcripts that will be used throughout the early stages of development prior to embryonic genome activation, a process known as ooplasm maturation. Furthermore, during follicular growth, epigenetic maturation occurs, which is essential for appropriate embryo development. We believe that transcripts and DNA methylation differ between blastocyst oocytes and those that cleaved but were arrested on day three. We devised a retrospective technique to identify transcripts in oocyte, cumulus, and granulosa cells, as well as DNA methylation connected with oocyte competence, in this work. We dissected and harvested ovarian follicles to achieve this purpose. We extracted and flash frozen the granulosa cells after rupturing them. The oocytes were put in maturation media droplets, and the cumulus cells and polar body were removed and kept the following day. To prevent spermatozoon interference, we chemically activated the oocytes and tracked their development (until they reached the blastocyst stage). We went back to their biopsies, cumulus cells, and polar bodies and did RNA-seq (biopsies and cumulus) and single polar body WGBS (polar bodies) when we collected the results 7 days later, i.e. 1-) embryos that cleaved but stopped development (termed CL) or 2-) embryos that cleaved and progressed until the blastocyst stage (termed BL). Additionally, after transcriptome results from oocyte-biopsy and cumulus cells, the granulosa cells from their individual oocytes were sequenced as a noninvasive strategy. This study is a follow-up to our previous work, "Assessment of Total Oocyte Transcripts Representation in bovine Using Single Ooplasm Biopsy with High Reliability." Following sequencing, we discovered that the two groups, BL x CL, were transcriptionally different in granulosa and biopsy samples, although cumulus cells were a poor predictor of oocyte competence. By analyzing the differentially expressed genes, we discovered multiple genes and pathways related to oocyte competency, demonstrating the efficacy of our method. Despite no change in morphology, these alterations in pathways and genes show that the oocyte CL group was transcriptionally and epigenetically delayed, with ferroptosis and necroptosis processes activated. The oocyte BL group demonstrated numerous molecular signaling, oocyte meiosis, GnRH signaling, G-protein cascade, and RNA stability pathways. In network analysis, we discovered GNAS, an imprinted gene and one of the most important essential genes. The transcripts from granulosa cells confirm the oocyte results. Nonetheless, transcriptional variations in granulosa cells were far greater than those in oocytes (97% x 34% variance), implying a completely distinct transcriptome in the follicular niche. In terms of the WGBS, we discovered differentially methylated areas linked with oocyte competency, as well as transcriptome results confirming the structures ability to predict outcome. These findings might be beneficial in clinical settings for those undergoing infertility therapy. In the oocyte, we discovered a complex transcriptional and epigenetic regulation network. Furthermore, mature cumulus transcription produced information that differed from the true content of the MII oocyte and granulosa cells before maturity. Our findings underscore the significance of maternal transcripts and epigenetic maturation in early parthenogenesis, as well as the use of granulosa cells as early indicators of competence.

genomics↗