3D-organization and spatial localization of chromatin and epigenetic marks linked to nucleolar activity in porcine...

Previous murine studies have established that large-scale chromatin modifications upon completion of oocyte growth are associated with nucleolar transcriptional silencing. These modifications seem essential both for completion of the oocyte's meiosis and subsequent embryonic developmental success. Investigating this putative interconnection between nucleolar transcriptional activity and spatial chromatin organization towards completion of oocyte growth in pigs, we scrutinized whether 3D chromatin organization and heterochromatin localization, along with epigenetic markers, could indicate oocyte quality and predict developmental competence of harvested porcine oocytes. Supravital brilliant-cresyl-blue (BCB) staining was used to classify porcine cumulus-oocyte-complexes (COCs) as fully grown (BCB+) or still growing (BCB-). Oocytes were analyzed via integrated 3D-immunofluorescence for nucleolar activity and heterochromatin markers, as well as 3D-DNA-FISH for specific heterochromatin sequences. Additionally, some oocytes were prepared for transmission electron microscopy (TEM). TEM revealed distinct ultrastructural differences between BCB+ and BCB- oocytes and validated BCB-staining as viable method for a rough assessment of oocyte developmental competence. Immunostaining identified all known germinal vesicle (GV) chromatin configurations (non-surrounded nucleolus (NSN), partially non-surrounded nucleolus (pNSN), partially surrounded nucleolus (pSN), surrounded nucleolus (SN)) and linked them to respective BCB categories. Nucleolar activity was detected only in NSN oocytes, predominantly from the BCB- group. Protein markers and FISH signals revealed significant 3D-organizational changes in chromatin between NSN and SN conformations, clustering around the nucleolus towards final oocyte maturation. These findings highlight an evident interconnection between nucleolar transcriptional silencing and specific 3D chromatin organization patterns, with changes in heterochromatin localization indicating completion of the oocytes' growth phase and marking higher competency for subsequent final maturation and embryonic development.