Slow and steady cell shrinkage reduces osmotic stress in bovine and murine oocyte and zygote vitrification

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Abstract
Does the use of a new cryoprotectant agent (CPA) exchange protocol designed to minimize osmotic stress improve oocyte or zygote vitrification by reducing sublethal cryodamage? The use of a new CPA exchange protocol made possible by automated microfluidics improved oocyte and zygote vitrification with superior morphology as indicated by a smoother cell surface, higher sphericity, higher cytoplasmic lipid retention, less cytoplasmic leakage and higher developmental competence compared with conventional methods. The use of more ‘steps’ of CPA exposure during the vitrification protocol increases cryosurvival and development in the bovine model. However, such an attempt to eliminate osmotic stress is limited by the practicality of performing numerous precise pipetting steps in a short amount of time. Murine meiotically competent germinal vesicle intact oocytes and zygotes were harvested from the antral follicles in ovaries and ampulla, respectively. Bovine ovaries were obtained from a local abattoir at random stages of the estrous cycle. A total of 110 murine oocytes, 802 murine zygotes and 52 bovine oocytes were used in this study. Microfluidic devices were fabricated using conventional photo- and soft-lithography. CPAs used were 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for equilibration solution and 15% EG, 15% DMSO and 0.5 M sucrose for vitrification solution. End-point analyses include mathematical modeling using Kedem–Katchalsky equations, morphometrics assessed by conventional and confocal microscopy, cytoplasmic lipid quantification by nile red staining, cytoplasmic leakage quantification by fluorescent dextran intercalation and developmental competence analysis by 96 h embryo culture and blastomere quantification. The automated microfluidics protocol decreased the shrinkage rate of the oocyte and zygote by 13.8 times over its manual pipetting alternative. Oocytes and zygotes with a lower shrinkage rate during CPA exposure experienced less osmotic stress resulting in better morphology, higher cell quality and improved developmental competence. This microfluidic procedure resulted in murine zygotes with a significantly smoother cell surface (P < 0.001), more spherical cellular morphology (P < 0.001), increased cytoplasmic lipid retention in vitrified and warmed bovine oocytes (P < 0.01), decreased membrane perforations and cytoplasmic leakage in CPA-exposed murine zygotes (P < 0.05) and improved developmental competence of vitrified and warmed murine zygotes (P < 0.05) than CPA exposure using the current clinically used manual pipetting method. It is necessary to design the microfluidic device to be more user-friendly for widespread use. The theory and approach of eliminating osmotic stress by decreasing shrinkage rate is complementary to the prevalent osmotic stress theory in cryobiology which focuses on a minimum cell volume at which the cells shrink. The auto-microfluidic protocol described here has immediate applications for improving animal and human oocyte, zygote and embryo cryopreservation. On a fundamental level, the clear demonstration that at the same minimum cell volume, cell shrinkage rate affects sublethal damage should be broadly useful for cryobiology. This project was funded by the National Institutes of Health and the University of Michigan Reproductive Sciences Program. The authors declare no conflicts of interest.

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