Biochemical changes in the cytoplasm of bovine oocytes during the in vitro maturation process: a Raman microscopy study.

The sequence and chronology of the main biochemical changes occurring in the cytoplasm of bovine oocytes during the in vitro maturation process were tracked by Raman microscopy applied to cells previously subjected to enzymatic digestion of the zona pellucida. Specific spectral markers for proteins, lipids and carbohydrates were used to evaluate the developmental status of the ooplasm at four different times. Spectral changes revealed that lipid accumulation was dominant during the first six hours of culture while protein content reached the average levels characteristic of mature oocytes within the last four hours of the maturation process. A time-dependent decrease in carbohydrates was also observed. Finally, the carbohydrate-to-protein (P1037/P1002) ratio proved to be sensitive enough to determine the cytoplasmic maturation state of bovine oocytes and promises to be useful in future research aimed at optimizing culture conditions through the promotion of protein accumulation in the ooplasm.

Exogenous activation and inhibition of plasminogen/plasmin activity during in vitro maturation of bovine cumulus-oocyte complexes: A biological and spectroscopic approach.

This study deals with the effect of plasminogen/plasmin on the in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs). Exogenous plasminogen activator streptokinase (SK) added to the IVM medium revealed similar values of cumulus expansion and oocyte nuclear maturation compared to controls (standard IVM medium). However, a decrease in both determinations was observed in COCs matured with the supplementation of ɛ-aminocaproic acid (ɛ-ACA), a specific plasmin inhibitor. After in vitro fertilization, no differences were observed in either cleavage or blastocyst rates between SK and control groups; however, ε-ACA treatment caused a decrease in both developmental rates. Zona pellucida (ZP) digestion time decreased in the SK group while it increased in the ε-ACA group. Raman microspectroscopy revealed an increase in the intensity of the band corresponding to the glycerol group of sialic acid in the ZP of oocytes matured with SK, whereas ZP spectra of oocytes treated with ɛ-ACA presented similarities with immature oocytes. The results indicate that although treatment with SK did not alter oocyte developmental competence, it induced modifications in the ZP of oocytes that could modify the folding of glycoproteins. Plasmin inhibition impairs oocyte maturation and has an impact on embryo development, thus evidencing the importance of this protease during IVM.