Integrative analysis of Smart-seq2 and DIA proteomics reveals dynamic formation of the Balbiani body during oocyte development in leopard coral grouper (Plectropomus leopardus)

Abstract

Abstract Background Membraneless RNA granules play important roles in germ cell development, yet their molecular composition and developmental dynamics remain poorly understood in fish. In this study, we investigated the Balbiani body (BB) and a nuage-associated nucleus-containing region (NNR) in oocytes of leopard coral grouper using laser capture microdissection (LCM), followed by integrated Smart-seq2 and DIA proteomics analyses. Because nuage is difficult to isolate reliably as a standalone compartment, the nucleus was co-dissected together with the surrounding nuage to generate the NNR samples. Results Ultrastructural observations showed that nuage appeared in oogonia and persisted throughout oocyte development, whereas the BB formed in primary oocytes and gradually expanded and dispersed into the cytoplasm. Integrative analysis of Smart-seq2 and DIA proteomics revealed relatively limited transcriptional variation among BB stages but more pronounced protein-level changes, while the NNR exhibited stage-associated molecular variation. The compartment-biased expression of established marker molecules was consistent with the known molecular characteristics of the BB and the presence of nuage-associated components in the NNR. However, functional signals detected in the NNR were interpreted as exploratory because of the contribution of nuclear molecules. Furthermore, Ddx6 and Igf2bp3 were identified and validated as candidate BB-associated markers. Their mRNA and protein signals corresponded to the BB region, and their stage-dependent localization closely paralleled BB formation, expansion, and dispersion during oocyte development. Conclusions These findings expand the candidate marker repertoire for the BB and provide a framework for investigating its developmental dynamics in fish oocytes. The molecular characteristics of the NNR provide preliminary information on the nuage-associated region, but their interpretation should remain exploratory because the dissected region contains the nucleus. Further studies using more specific approaches to isolate nuage will be required to clarify its molecular composition and its possible relationship with the BB.

The authors' abstract, as published at the source. BMC Genomics, 2026 · DOI ↗