(6) Differential Activation of Extracellular Vesicle Machinery Genes in Bovine Oocytes and Preimplantation embryos

Mohamed O. Taqi¹, Dessie Salilew-Wondim^2,3, Ernst Tholen², Michael Hoelker³, Franca Rings², Karl Schellander² and Dawit Tesfaye⁴

¹Central Laboratory for Agricultural Climate, Agricultural Research Center, Giza, Egypt

²Institute of Animal Science, Animal Breeding and Husbandry group, University of Bonn, Germany

³Department of Animal Science, Biotechnology and Reproduction of Farm Animals, University of Goettingen,

Goettingen, Germany

⁴Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory (ARBL), Colorado State University, Fort Collins, CO, USA

Abstract Text:

Extracellular vesicles (EVs) have emerged as pivotal mediators in intercellular communication, with significant potential in diagnostics and therapeutic delivery. However, the regulatory mechanisms governing their biogenesis and transport remains poorly understood, particularly in bovine reproductive processes. Therefore, this study aims to elucidate the expression dynamics of key genes involved in EV biogenesis (STAM1, TSG101, ALIX, VPS4, TSAP6) and transport (RAB11, RAB35, RAB27a, RAB27b) during bovine oocyte maturation and early embryo development. To investigate this, bovine ovaries were collected from a slaughterhouse, and cumulus-oocyte complexes (COCs) were aspirated from healthy small follicles (2-8 mm). While triplicate biological pools of immature oocytes (IO, n = 50 per pool) and cumulus cells (ICCs) were directly snap-frozen, subpopulation of COCs underwent in vitro maturation (IVM) and in vitro culture (IVC) following in vitro fertilization (IVF). Presumptive zygotes were cultured under 5% oxygen, with triplicates of sample pools collected for downstream analysis at 2-cell (n = 20 per pool), 4-cell (n = 20 per pool), morula (n = 15 per pool), and blastocyst (n = 10 per pool) stages. Triplicate pools of mature oocytes (MO, n = 50 per pool) and the corresponding cumulus cells (MCCs) were also used for downstream analysis.  RNA from different stages of oocytes and embryos was isolated from all samples using the PicoPure™ RNA Isolation Kit and then converted to cDNA for quantitative gene expression analysis. Gene specific primers were used to quantify their expression using iTaq™ Universal SYBR^® Green Supermix (Bio-Rad Laboratories GmbH, München, Germany) in Applied Biosystem® StepOnePlus™ (Applied biosystems, Foster City, CA, USA). The mRNA expression data were analyzed using comparative Ct (2^−ΔΔCt) methods and the geometric mean of the expression level of ACTB and GAPDH was used for normalization. Data analysis was performed using least three independent replicates. Data were statistically analyzed with Student’s two-tailed t test for oocyte and cumulus cell comparisons, and one-way ANOVA with Tukey’s post hoc test for embryonic stages using GraphPad Prism software version 7 (GraphPad Software, La Jolla, California, USA). The difference between means was considered significant when P value ≤ 0.05. The quantitative analysis revealed significant upregulation of TSG101 and RAB27a mRNA levels in MO compared to IO. In MCCs versus ICCs, all investigated biogenesis-related genes (STAM1, TSG101, ALIX, VPS4, TSAP6) was significantly upregulated by 2-3-fold, while transport-related genes exhibited divergent expression patterns: RAB27b mRNA increased by 4-fold whereas RAB11 expression decreased. During embryogenesis, most biogenesis and transport genes were downregulated from the 2-cell to blastocyst stages relative to 2-cell stage. However, the expression of TSAP6 and RAB11 were exceptions, which were upregulated by up to 31-fold in the morula stage and then decreased by approximately half at the blastocyst stage. These findings suggest cell- and stage-specific regulation of EV machinery genes, with pronounced activity during oocyte maturation and early embryonic cleavage stages. Further exploration of EVs release dynamics during these critical stages could enhance our understanding of the role of EVs in embryo-maternal interaction during pregnancy and advance their use in therapeutic and gene delivery strategies.