Change in the ability of granulosa cells to elongate transzonal projections during follicle development

Cumulus cells (CCs) and mural granulosa cells (MGCs) have different phenotypes and different roles in the follicle. However, our previous reports have suggested that CCs and MGCs from bovine early antral follicles become MGC-like cells and CC-like cells, respectively. In this study, we examined whether granulosa cells (GCs) from secondary follicles and MGCs from late antral follicles were able to reconstruct complexes with transzonal projection (TZP)-free denuded oocytes (DOs) and regenerate TZPs similar to MGCs from early antral follicles. Furthermore, to confirm that the regenerated TZPs were functional, the development of reconstructed complexes and oocyte growth in the complexes were determined. After coculture, GCs were able to reconstruct complexes with DOs and regenerate TZPs similar to MGCs from early antral follicles. In addition, the oocytes in integrally reconstructed complexes grew fully and acquired meiotic competence, suggesting that the regenerated TZPs are functional. In contrast, MGCs from late antral follicles had lost the ability to elongate TZPs. Since the ability to regenerate TZPs differed among the cells, the transcriptome was analyzed among GCs, CCs, and MGCs collected from follicles of different sizes. The characteristic of TZP generation coincided with transcriptome changes in two directions from GCs to CCs and MGCs. In conclusion, until the early antral follicle stage, bovine GCs, CCs, and MGCs have common characteristics to elongate TZPs and form antrum-like structures to support oocyte growth in vitro. Furthermore, as the follicle develops, MGCs lose the ability to elongate TZPs. Overall design: We investigated whether GCs from secondary follicles and MGCs from late antral follicles were able to reconstruct complexes with TZP-free denuded oocytes and regenerate TZPs similar to MGCs from early antral follicles using in vitro growth culture. To confirm that the characteristic of TZP generation was coincident with transcriptome changes, we performed gene expression analysis using data obtained from RNA-seq of 5 different cells at different size follicles.