Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 °C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

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In the mouse, the use of the DNA-binding fluorochrome Hoechst 33342 allows the classification of fully-grown antral oocytes into two categories distinguished by their chromatin conformation: surrounding nucleolus (SN) and not-surrounding nucleolus (NSN) oocytes, the former capable of completing development, the latter unable to proceed beyond the 2-cell stage. In the present study, time-lapse observation of SN and NSN oocyte GV-to-MII transition highlighted differences in the timing of germinal vesicle breakdown (GVBD) and polar body I (PB-I) extrusion. PB-I extrusion, but not GVBD, revealed the presence of three main groups of significantly different oocytes: Group A (456-576 min) comprising mainly SN oocytes (91.4%), group B (584-728 min) entailing an almost equivalent percentage of SN (52.7%) and NSN (47.3%) oocytes, whereas group C (736-896 min) consisting of almost all NSN (94.4%) oocytes. In a further set of time-lapse experiments, GV oocytes were in vitro matured without Hoechst staining and, depending on the timing of PB-I extrusion, sorted into group A, B or C, inseminated with sperm and observed throughout preimplantation. The results show that 26.2 ± 12.3% of group A, 2.4 ± 5.0% of group B and none of group C MII oocytes developed to blastocyst. Overall, this study shows that SN oocytes that complete MI earlier are those with a better developmental competence. The possibility to avoid the use of the invasive DNA-binding fluorochrome Hoechst is relevant for future applications in human and domestic animal reproductive technologies.

In the ovary, acquisition of oocyte developmental competence depends on a bidirectional exchange between the gamete and its companion cumulus cells (CCs). In this study we investigated the contribution of CCs surrounding oocytes of known developmental competence or incompetence to the acquisition of oocyte developmental competence. To this end, feeder layers of CCs (FL-CCs) were prepared using CCs isolated either from: (1) developmentally competent mouse oocytes whose nucleolus was surrounded by a chromatin ring (FL-SN-CCs); or (2) developmentally incompetent mouse oocytes whose nucleolus was not surrounded by a chromatin ring (FL-NSN-CCs). Denuded, fully grown oocytes (DOs) were matured to the MII stage on either FL-SN-CCs or FL-NSN-CCs, inseminated with spermatozoa and cultured throughout preimplantation development. FL-SN-CCs significantly improved the acquisition of oocyte developmental competence, with a blastocyst development rate equal to that for maturation of intact cumulus–oocyte–complexes. In contrast, DOs matured on FL-NSN-CCs or in the absence of CCs exhibited developmental failure, with embryos arresting at either the 4-cell or morula stage. These results set a culture platform to further improve the protocols for the maturation of DOs and to unravel the molecules involved in the cross-talk between the gamete and its companion CCs during the germinal vesicle to MII transition.

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