NOBOX, a marker of oocyte developmental competence

Introduction NOBOX is an oocyte transcription factor with a crucial role during folliculogenesis: lack of this protein results in the failed progression of oocyte growth beyond the primordial follicle stage. Although the presence and function of NOBOX in the early stages of folliculogenesis is clear, a detailed description of its expression beyond the primordial stage is still lacking. Here, we report the expression of both the Nobox transcript and protein during oocyte growth. Materials and methods A detailed description of the protocols used is given in Belli et al. (2013). Briefly, following a collagenase and mechanical treatment, oocytes were isolated from ovaries of 3-4 week-old B6C3F1 mice and classified based on their size (six classes: 10-30, 31-40, 41-50, 51-60, 61-70, 71-80 μm in diameter) and chromatin organisation (NSN, Non Surrounded Nucleolus or SN, Surrounded Nucleolus). The chromatin organization, observed with the supravital fluorochrome Hoechst 33342, is a morphological marker that, when used with fully-grown antral oocytes allows the identification of developmentally competent (SN) vs. incompetent (NSN) oocytes. At least 30 oocytes from each class were collected, for both gene (Real time-PCR) or protein (immunofluorescence) expression analysis. Results Nobox transcripts are present in both SN and NSN gametes throughout oocyte growth and at the MII stage derived from in vitro grown NSN (MIINSN) and SN (MIISN) fully-grown antral oocytes. Ovulated MII oocytes and MIISN have similar Nobox expression, significantly lower than that of MIINSN oocytes. NOBOX protein is confined in the nucleus of both 10-60 μm NSN and SN oocytes. Then, from 61-70 μm, whilst NSN oocytes continue to display NOBOX localisation, SN oocytes show an abrupt down-regulation of this protein, which is almost undetectable, except for few perinucleolar areas. This down-regulation is conserved also in oocytes of 71-80 μm in diameter. In this latter class, we observed also the presence of oocytes (about 21%) with a lower NOBOX immunofluorescence and a chromatin organization intermediate between that of the SN and NSN type. Following meiotic resumption, MIINSN oocytes show strong NOBOX localization within the whole cell volume, likely as a consequence of its diffusion after GVBD; whereas in MIISN and ovulated MII oocytes NOBOX is down-regulated. Conclusions The sharp difference of NOBOX expression in developmentally competent (SN, down-regulated) and incompetent (NSN, up-regulated) oocytes makes this protein a putative marker of their quality.