Spheroid granulosa cell supplementation improves granulosa cell–oocyte interactions and enhances in vitro growth of oocytes in cows

Oocytes develop through bidirectional communication with surrounding granulosa cells (GCs), and differences in the GC environment represent a major distinction between in vivo and in vitro oocyte growth systems. However, the influence of GC number and organization on oocyte growth and quality during in vitro culture remains unclear. In this study, oocyte–granulosa cell complexes (OGCs) derived from early antral follicles (0.5–0.7 mm) of cows were supplemented with spheroid granulosa cells (GC-S) derived from antral follicles (3–5 mm). GC-S were incorporated into OGCs during 14 days of incubation. Oocyte characteristics, maturation, and fertilization competence were evaluated, and the transcriptomic profiles of surrounding GCs were analyzed by RNA sequencing. GC-S supplementation increased transzonal projections and improved GC survival within OGCs, indicating enhanced GC-oocyte interactions. It also increased ATP content, oocyte diameter, and DNA methylation levels, reaching levels comparable to those observed in in vivo-grown oocytes. Furthermore, GC-S supplementation significantly improved oocyte maturation and fertilization rates, although these remained lower than those of in vivo counterparts. In contrast, lipid content, mitochondrial DNA copy number, and mitochondrial membrane potential were not affected and remained lower than those of in vivo-grown oocytes. RNA-seq analysis revealed that GC-S supplementation altered the expression of genes associated with focal adhesion and cytoskeleton-related pathways toward those observed in vivo. These results demonstrate that spheroid GC supplementation improves GC-oocyte interactions and partially restores in vivo-like characteristics of oocytes during in vitro growth.