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Integrated Analysis of Granulosa Cells and Extracellular Vesicles Reveals Predictive Biomarkers of Oocyte Maturation in the Southern White Rhinoceros

ABSTRACT: Assisted reproductive technologies (ARTs) are an imperative conservation tool to rescue species from extinction. An essential step towards technological advancement and ARTs’ success is identifying optimal in vitro culture conditions to mature oocytes, carry out fertilization, and culture embryos for future transfer. The role of extracellular vesicles (EVs) and granulosa cells (GC) in follicular development, oocyte maturation, and fertilization has been extensively studied for decades in human and domestic species models. This study aimed to characterize transcriptomic differences in GC and evaluate the communication between EVs in the follicular fluid and GC miRNA cargo between oocytes that matured (PB) and those that did not mature (NoPB) after in vitro culture. Overall, in GC, 1,117 transcripts (annotated and unannotated) were differentially expressed between groups. In the miRNA from the GC, 11 were differentially expressed between groups, all higher in the PB group. For the miRNA in follicular fluid EVs, there were 15 miRNA differentially expressed. When comparing targets of the miRNA identified to the differentially expressed genes, multiple genes were being targeted and affected. These miRNA and genes are potential targets for further investigation to be used for support during in vitro maturation or to develop markers for an oocytes potential to mature during in vitro maturation. In conclusion, extracellular vesicle–mediated molecular signaling during folliculogenesis in the SWR may offer critical insights into physiological and pathological reproductive conditions that contribute to abnormal fertility, ultimately informing the development of novel reproductive strategies to enhance ART outcomes in this species and others.

ORGANISM(S): Ceratotherium simum simum

PROVIDER: GSE326340 | GEO | 2026/07/29

REPOSITORIES: GEO

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Project description:In mammals, the capacity of the female germ cell, the oocyte, to develop into embryo is acquired throughout meiotic maturation. Immature oocyte cannot be fertilized while mature oocyte is apt to accept spermatozoa and to develop an embryo. In a follicle, the oocyte is surrounded by mural granulosa cells (GC) and is physically and metabolically coupled with specialized granulosa cumulus cells (CC) which play an important role in oocyte maturation and fertilization. Factors expressing in GC and CC during maturation may reflect the oocyte quality, i.e. its capacity to be fertilized and assure early embryo development. However, the modifications of the content and the amount of peptide/proteins in the oocyte and the surrounding CC during oocyte maturation are mostly unknown and so there is not an accurate way of evaluating/monitoring how different in vitro maturation (IVM) protocols being in use in assisted reproduction technologies, can affect the process. In this context, Intact Cell MALDI-TOF Mass Spectrometry (ICM-MS) was applied to bovine follicular cells (bovine single oocytes, cumulus cells and granulosa cells) in order to characterize proteomic changes that occur in the follicle during female gamete development. In order to characterize finely endogenous molecular species observed on ICM-MS profiles and to identify markers of interest with their post-translational modifications, we carried out top-down proteomic on the different follicular cells from oocyte, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts. Prior to top-down MS using a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos, depending on the amount of available biological material, we employed three analytic strategies as a direct infusion, a mono-dimensional liquid chromatography (µLC-1D-MS/MS) and an off-line multi-dimensional liquid chromatography combining four fractionations (based on reverse phase or gel filtration LC) to µLC-MS/MS. Here, we deposited our dataset from µLC-1D-MS/MS (analyses of oocytes, oocyte-cumulus complexes, cumulus cells and granulosa cells protein extracts) and MDLC-MS/MS (analyses of granulosa cells protein extract).

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Integrated Analysis of Granulosa Cells and Extracellular Vesicles Reveals Predictive Biomarkers of Oocyte Maturation in the Southern White Rhinoceros

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