26 The role of granulosa cells and extracellular vesicles in the acquisition of oocyte competence in the southern white rhinoceros

An appropriate intrafollicular environment supports the acquisition of developmental competence in oocytes. Extracellular vesicles (EVs) participation in the metabolic exchanges between ovarian follicle cells and the enclosed oocyte are crucial elements in this developmental process. Transcriptomic profiling of follicular cells, such as granulosa cells (GCs), can provide biomarkers for oocyte competence. Understanding the communication between these two fundamental aspects (GCs and oocytes through EVs) of oocyte development will ultimately improve assisted reproductive technologies (ARTs) in the southern white rhinoceros (SWR).This study aimed to (1) identify RNA transcripts from in vivo GCs to assess oocyte competence after IVM and (2) identify EVs in the corresponding follicular fluid (FF) that led to mature or immature oocytes after IVM. For the first aim, in vivo mural GCs were collected from two follicles after ovum pickup (OPU) that resulted in meiotically competent oocytes following IVM and two follicles that resulted in oocytes failing to mature. Oocyte meiotic competence was determined through the visualization of an extruded polar body following IVM. Total RNA from GCs was isolated, and cDNA libraries were prepared and sequenced on a NovaSeq 6000. Reads were aligned to CerSimCot1.0 using HISAT2, and FeatureCounts was used for read counts. For the second aim, EVs were isolated from the FF of the four follicles using consecutive rounds of ultracentrifugation followed by size exclusion chromatography. EVs were assessed for particle size distribution, concentration, morphological structure, and biochemical alterations.Overall, 16 754 transcripts were identified in GCs, regardless of the maturation status of oocytes. A total of 897 differentially expressed genes (DEGs) were interconnected to oocyte maturation status. Of these DEGs, 460 were upregulated in oocytes that matured following IVM and 437 in oocytes failing to mature. In addition, 2825 genes were found to be expressed exclusively in GCs from oocytes that matured following IVM, and 3909 genes in GCs from those that did not mature. EVs exhibited typical cup-shaped morphology. Nano-tracking analysis demonstrated the presence of primarily small vesicles in the expected size range for exosomes (30–150 nm), but there was a difference in average size between EVs from FF from oocytes that matured (144.8 nm) and those from FF with oocytes that did not mature (139.2 nm). There was a relatively similar concentration of EVs between follicle groups (2.6 × 10⁹ ± 0.08). Immunoblotting confirmed the presence of EV-specific proteins CD63 and TSG101 and the absence of mitochondrial cellular contaminant cytochrome C. This study provides preliminary insights into oocyte competence-dependent integrative transcriptomic profiling of rhinoceros GCs. Future experiments to investigate rhinoceros EV-miRNAs shed into the FF of mature versus immature oocytes will elucidate the regulatory function and significance of their role in the governing oocyte function.