Time-lapse imaging of chromatin and cytoplasmic movements occurring during the GV-to-MII transition: in search for markers of mouse oocytes developmental competence

The transition from the germinal vesicle to the metaphase II stage (GV-to-MII transition) is crucial to the acquisition of the oocyte developmental competence. Here, using live, time-lapse, imaging we describe the movements occurring during the GV-to-MII transition to the chromatin (CHR-MOV) and to the cytoplasm (CYTO-MOV) of mouse oocytes of known developmental competence or incompetence. Fully-grown cumulus-oocyte-complexes were punctured from the ovarian surface, the GV oocytes isolated and stained with the supravital Hoechst 33342 fluorochrome (Ho) which allowed the identification of gametes whose nucleolus is surrounded by a ring of Ho-positive chromatin (surrounded nucleolus, SN, oocytes) from those that lack this ring (not surrounded nucleolus, NSN, oocytes). Importantly, when in vitro cultured to MII and inseminated with sperm, whilst SN oocytes may develop to term, NSN oocytes arrest development at the 2-cell stage. The time-lapse observation of CHR-MOV describe distinct chromatin changes in NSN compared to SN oocytes, with a longer GV-to-MII transition in NSN oocytes that reach the M-phase without the gathering of heterochromatin regions around the nucleolus. Furthermore, by coupling bright-field time-lapse observations with the Particle Image Velocimetry method, we analysed the CYTO-MOV of these two types of oocytes. We showed that SN and NSN oocytes exhibit distinct profiles and, at four main time-frame intervals, their CYTO-MOV velocity is significantly different. In addition, we integrated the information of the CYTO-MOV profile of each single oocyte with an artificial neural network analysis that blindly identified the oocyte as SN or NSN with a robust probability. The presence of SN and NSN oocytes in all mammals, including humans, extends the interest of these results to the field of assisted reproductive technologies (ART).