Organelle morphodynamics in human mature oocytes after cryopreservation. Ultrastructural analysis at different time intervals during thawing

Introduction: During freeze-thawing, the human metaphase II (MII) oocyte is exposed to a variety of physical and chemical conditions that may endanger its competence to fertilization and even its mere survival. In this study we evaluated presence and amount of: a) ooplasmic vacuolization, b) organelle-specific associations such as mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and mitochondriavesicle (MV) complexes, and c) cortical granules (CGs). Materials and Methods: MII oocytes were subjected to slow freezing through two-step propanediol (PrOH) dehydration with 0.75-1.5 mol/l PrOH and 0.2 mol/l sucrose and examined by light and transmission electron microscopy (TEM) at different time intervals during thawing. Cryopreserved oocytes were fixed after being transferred in 1.0 mol/l PrOH and 0.3 mol/l sucrose (group A, n=15), 0.5 mol/l PrOH and 0.3 mol/l sucrose (group B, n=15) and 0.3 mol/l sucrose (group C, n=15). Fresh MII oocytes (n=15) were used as controls. Results: Morphometric and TEM analysis revealed that vacuoles were only occasionally detected in the ooplasm of 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. M-SER aggregates, large and abundant in the ooplasm of fresh controls, significantly decreased in number following freezing, particularly in the oocytes belonging to groups A and B. MV complexes were instead small and scarce in fresh control oocytes 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. CGs was scarce in all cryopreserved oocytes in respect to those found in fresh controls and gradually diminished as thawing progressed. 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 when the lowest concentration of PrOH is reached. Significant variations in the number of M-SER aggregates and MV complexes occurred during the 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. It seems also worth noting that all systems of 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 the thawing process.