Evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model

Interest in oocyte cryopreservation has recently increased. Cattle oocytes are sensitive to low temperatures, and despite the efforts of numerous research groups cryopreservation of oocytes remains a difficult task. This problem may be in part due to the large size of bovine oocytes, which consequently have a low surface to volume ratio, making it more difficult for water and cryoprotectants (CP) to move across the cell plasma membranes. Several attempts to improve the survival rate of oocytes pioneered the establishment of different cryopreservation techniques. Recently, vitrification (based on direct plugging of samples into liquid nitrogen) has been used for the cryopreservation of oocytes as an alternative technique over traditional slow-rate freezing method. One of the most successful ultrarapid vitrification techniques is the Cryotop vitrification (CTV) that has resulted in excellent survival and developmental rates with human and bovine Metaphase II (MII) oocytes. However, despite the improvement in survival and cleavage rates, development into blastocysts is still poor, probably because ultrastructural and physiological changes that occur as a consequence of vitrification. For this reason, the aim of this study was the evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model. It is known that vitrification causes several ultrastructural and structural alterations, including damages of meiotic spindle apparatus in oocyte of several species. So, the starting point of this work was the evaluation of structural changes of in vitro matured bovine oocytes following cryopreservation and simple exposure to CPs (toxicity test). The first conclusion of this experiment was that the simple exposure of oocytes to CPs causes damages to the meiotic spindle and chromosome organization similar to those induced by the whole vitrification protocol. The second conclusion, according to our opinion, was even the most interesting: we observed the occurrence of spontaneous parthenogenetic activation, indicated by the highest presence of oocytes in Telophase II stage (TII) in the oocytes of toxicity group compared to vitrification group. We speculate that the lower activation observed in the vitrification group may be referred to the slowing down of the metabolic activity subsequent to thermal shock, and hence that activation after vitrification may occur later than 2 h post-warming. Restore period of oocytes after vitrification (2 h) coincide with the moment where we normally would perform the intervention of in vitro fertilization (IVF) because it is believed that during this period of time there is a restore of normal morphology of the spindle. To confirm this hypothesis, nuclear stage evaluation was carried out in bovine oocytes fixed and dye at later times post-warming, and precisely: 2 (time 2) and 4 h (time 4) post-warming. In addition, in order to comprehend the mechanism inducing the spontaneous activation and, hence to hypothesize a corrective strategy, an additional objective was to measure the activity of the Maturation-Promoting Factor (MPF) and the Mitogen- Activated Protein Kinase (MAPK) in fresh, exposed to CPs and vitrified bovine oocytes. Results of this evaluation showed that the incidence of activation in the toxicity and vitrification groups enhanced from T2 to T4 and the phenomenon was more evident at T4. These values supports our hypothesis referred to the slowing down of the metabolic activity subsequent to thermal shock in oocytes of vitrification group, confirming that activation after vitrification may occur later than 2 h post-warming. These data were confirmed even from the MPF and MAPK levels measurement: the activity of MPF decreased in oocytes of vitrified and exposed groups compared to the control, which reflected the differences in parthenogenetic activation rates. In particular, we showed that vitrification determined in bovine oocytes a fall in MPF activity, which was evident since 2 h post-warming and was not restored at 4 h post-warming. A similar pattern was found in vitrified oocytes for the MAPK activities. after 2 and 4 h, both in vitrification then in toxicity group there is a decrease of MPF and these levels were similar. A possible method for reduce the percentage of parthenogenetic activation after oocytes vitrification could be the treatment of the oocytes with substances able to stabilize the action of MPF and MAPK. Theoretically, the stabilization of these molecules could avoid these adverse effects. It has been reported that molecules such as caffeine could restore MPF activity in oocytes of different species. For this purpose, MII bovine oocytes were treated with different concentration of caffeine (0, 10, 20 and 40 mM) in in vitro maturation (IVM) medium before and after vitrification in order to evaluate the effect on MPF and MAPK activity, oocyte spontaneous parthenogenetic activation and embryo development after the treatment. Unfortunately, the entirety of these results demonstrated that the caffeine treatments of the oocytes, at the three concentrations tested fail to improve the vitrification efficiency in cattle. Indeed, caffeine at the lower concentrations, i.e 10 and 20 mM, did not increase the MPF and MAPK activities and hence did not reduce spontaneous activation. Subsequently, no improvement was recorded in terms of cleavage and blastocyst rates following IVF and IVC in both groups. Furthermore, the only concentration (40 mM) that actually increased the kinases and reduced the spontaneous activation rates was also toxic for the oocytes, as shown by the high incidence of condensed chromatin, the low percentage of oocytes with a normal MII configuration and low cleavage and blastocyst rates. Oocytes that survive cryopreservation may also accumulate reactive oxygen species (ROS). ROS are known to exert harmful effects such as mitochondrial damage, adenosine triphosphate (ATP) depletion, altered calcium oscillation during fertilization, apoptosis, and developmental block. Consequently, developmental ability of cryopreserved oocytes and embryos may be compromised. Therefore, another aim of this work was designed to investigate the effect of vitrification and CPs exposure on ROS activity of bovine in vitro matured oocytes.The results of this experiment showed an increase of ROS levels following both vitrification and exposure to CPs compared to the control, although the difference was not statistically different. The most evident result was that the ROS levels in fresh control oocytes were stable, whereas a high variability among replicates was recorded in both vitrified and exposed oocytes. These results indicate that one of the damages of bovine oocytes following CTV vitrification is the increased ROS levels, that may be responsible of the poor developmental potentials. A new approach to improve the cryotolerance and developmental competence of gametes and embryos is to expose them to sub lethal stress before manipulation. This exposure to sub lethal stess can increase the subsequent stress tolerance, morphological intactness and, presumably, cryopreservation ability. This stress can be osmotic, using non permeating solutes such as NaCl, or thermic, using high temperature. To explore the effect of NaCl in improving the cryotolerance and developmental competence of bovine oocytes, oocytes were incubated with different concentration of NaCl (0 M, 0.1 M, 0.15 M and 0.2 M) before vitrification. The results of this experiment demonstrated that a short exposure to 0.15 M NaCl prior to vitrification improves the cryotolerance of in vitro matured bovine oocytes, as indicated by increased survival and cleavage rates after IVF of vitrified-warmed oocytes. Furthermore, the percentage of cleaved oocytes reaching the blastocyst stage also tended to increase. The second type of stress, heat shock, was applied to oocytes at different period of IVM: after 13h of IVM at 39°C for 9h at 42°C; after 16h of IVM for 6h at 42°C; after 19h of IVM at 39°C for 3h at 42°C. The results of this experiment shown that, a brief (3 h) exposure of oocytes to elevated temperatures prior to vitrification improves their cryotolerance, as shown by the increased cleavage rate, whereas a more prolonged exposure to elevated temperatures is detrimental. In conclusion, regardless for the necessity of other studies, the results of this series of experiments presented the different kind of oocytes cryopreservation problems and try to present a series of solution or suggestions to improve this very important technique for the bovine model.