Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine
Authors: Ambrizi Dewison Ricardo , Camara De Bem Tiago Henrique , Nociti Ricardo Perecin , Puttini Paixao Joao Vitor , Chiaratti Marcos Roberto , Sangalli Juliano Ricardo , Da Silveira Juliano Coelho , Perecin Felipe , de Mattos Oliveira Elisangela Chicaroni , Sterman Ferraz Jose Bento , Therrien Jacinthe , Smith Lawrence Charles , Meirelles Flavio Vieira 
Published: April 2023

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 discoveredGNAS, 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% × 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 structure’s 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.