Ultrastructural modifications in the organular pattern of human metaphase II oocytes subjected to freeze-thawing

Human metaphase II (MII) oocyte is very susceptible to cryodamage. In this study we evaluated the modifications occurring in the organular pattern of human MII oocytes subjected to freeze-thawing and examined by light and transmission electron microscopy (TEM) at different time intervals during thawing (beginning, group A; middle, group B; end, group C). Fresh MII oocytes were used as controls. Morphometric and TEM analysis revealed that vacuoles were only occasionally detected in fresh controls. Conversely, vacuoles were numerous in the cryopreserved oocytes of group A and appeared to reach an even larger number in group B oocytes. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates, large and abundant in fresh controls, significantly decreased in number following freezing, particularly in the oocytes belonging to groups A and B. Mitochondria-vesicle (MV) complexes were instead small and scarce in fresh controls but augmented after freezing, being especially abundant in the oocytes belonging to group B. Vacuoles and MV complexes both diminished in the oocytes belonging to group C, whereas M-SER aggregates increased in number. Cortical granules (CGs) were scarce in all cryopreserved oocytes in respect to those found in fresh controls and gradually diminished as thawing progressed. In conclusion, this study proves that vacuoles, generally regarded as markers of oocyte cryodamage during slow cooling, may form during freezing, but become numerous during thawing, particularly in the middle phase. Significant variations in the number of M-SER aggregates and MV complexes occurred during freeze-thawing, suggesting a dynamic process of transition between these two forms of organelle associations. This study also evidences that a premature CG exocytosis progressively occurs during the whole freeze-thawing procedure. Thus, all ooplasmic membranes appear significantly concerned by freeze-thawing but, except for CGs, their alterations seem to undergo a partial or, more rarely, an almost complete recovery at the end of thawing.