Chromatin organization, spatial localization of heterochromatin sequences and nucleolar activity, change towards final maturation in porcine oocytes - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement Access content directly
Conference Poster Year : 2023

Chromatin organization, spatial localization of heterochromatin sequences and nucleolar activity, change towards final maturation in porcine oocytes

Abstract

The chromatin conformation of oocytes is subjected to large-scale modifications correlated to transcriptional silencing during final maturation. These modifications seem essential both for completion of the meiosis and subsequent embryonic developmental success. However, in pig, knowledge about spatial heterochromatin organization and nucleolar transcriptional activity toward the completion of oocyte growth are still lacking. In the present study, 300 porcine cumulus-oocyte-complexes (COCs) were recovered from ovaries and then divided into 2 groups, pre-categorized by supravital brilliant-cresyl-blue (BCB) staining, defining either fully mature (BCB+) and still maturating (BCB-) oocytes. Three dimension nuclear organization of heterochromatin was determined by either 3D-immunofluorescence (n=140) using specific antibodies against H3K9me3 and centromeric proteins (CENPs) and by 3D-DNA-FISH experiments (n=140) using fluorescent oligonucleotides specific for porcine meta- and acrocentric chromosomes. Nucleolar activity was detected using antibodies upstream binding factor (UBF). All images were acquired using laser scanning confocal microscpy. Finally, oocytes (n=20) were prepared for TEM according to standard protocol. Qualitative assessment of cellular ultrastructure by TEM revealed distinct differences between BCB+ and BCB- oocytes and supported BCB-staining as viable method for rough categorization regarding maturation status. All chromatin conformations were detected: from non-surrounded nucleolus (NSN) to surrounded nucleolus (SN) and their intermediate conformations (pNSN, pSN) in both BCB groups. However, the BCB+ group contained a higher percentage of oocytes expressing chromatin conformations categorized as mature (pSN and SN), whereas the opposite was true for the BCB- group. UBF-activity was only present in NSN and pNSN categorized oocytes and detected significantly more often in the BCB- group. The distribution of centromeric (CENP) and pericentromeric chromatin (H3K9me3) as well as repeated sequences DNA-FISH signal displayed distinct changes in their 3D-organization between NSN and SN conformation, characterized by significant signal-condensation around the nucleolus in SN oocytes. Altogether, these results indicate that oocytes from the BCB+ group, i.e. oocytes with higher competency for embryonic development, display specific heterochromatin organization patterns related to final oocyte maturation.
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Dates and versions

hal-04356806 , version 1 (20-12-2023)

Identifiers

  • HAL Id : hal-04356806 , version 1

Cite

Merle Friederike Fenner, Michal Benc, Alexandra Bartkova, Maria Pihl, Martine Chebrout, et al.. Chromatin organization, spatial localization of heterochromatin sequences and nucleolar activity, change towards final maturation in porcine oocytes. SSR Annual Meeting, Reproductive Sciences: From innovation to impact, Jul 2023, Ottawa, Canada. ⟨hal-04356806⟩
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