Dry Preservation of Germinal Vesicles: Development of a New Formulation to Preserve DNA Integrity Under Adverse Environmental Conditions
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Abstract
Advances in the cryopreservation of genetic material has revolutionized human reproductive medicine and animal conservation efforts. Despite many successes, oocytes have been more challenging to cryopreserve than sperm. Their large size makes it difficult to load cryoprotective agents (CPAs) into the cell without toxicity issues, and the high water content can cause damage from formation of intracellular ice crystals during cooling and rewarming. Research has shown that preservation of the genetic material can be achieved with fewer preservation challenges if the germinal vesicles (GVs) are targeted for preservation instead of the whole oocyte. The gamete can then later be reconstituted by depositing the preserved DNA into an enucleated cell. The current study evaluated a novel preservation composition to enable dry preservation of these GVs, which eliminates the need for cryogens, refrigerated transport and storage, and the use of toxic cryoprotectants. The compositions evaluated were based on the naturally occurring protectant trehalose. Trehalose is a sugar that is naturally occurring in animal species, called anhydrobiotes, that can survive extreme dehydration and other adverse conditions. Trehalose is also used in the pharmaceutical industry for its ability to form a glass under appropriate conditions. The glassy state is a solid with an amorphous conformation that is reached by drying (or cooling) the solution in a manner that prevents molecular ordering and crystal formation. Molecular mobility is practically halted in the glassy state, which prevents degradation of materials that are embedded in such a glass. This glassy state must be stringently maintained in order to maintain functionality of preserved materials, and in the specific case of trehalose, samples held above 44% Relative humidity (RH) will quickly crystallize. The current study investigated additives that could enable more flexible storage and shipping conditions, especially high humidity environments. Feline oocytes were isolated, denuded to separate the oocyte from surrounding cells, permeabilized to allow for intracellular loading of the preservation solution, then immediately dehydrated to an amorphous solid state in an 11% RH environment using microwave-assisted drying to reach this glassy state. These preservation solutions included the disaccharide sugar trehalose, along with salt additives of the choline family, specifically choline citrate, choline acetate, and choline chloride. Choline salts are naturally occurring electrolytes that have been observed to delay or prevent crystallization in trehalose glass systems in previous work. The dried, porated oocytes were subjected to storage conditions simulating ideal (11% RH) and adverse (76% RH) conditions, then rehydrated and analyzed for recovery fraction and DNA fragmentation by TUNEL analysis. High RH is known to cause crystallization in trehalose solutions and can possibly contribute to cellular damage. The formation of crystals in preserved cells can also potentially cause DNA fragmentation. Compositions with salt additives were shown to moderately increase the recovery fraction of GVs from the storage substrate after exposure to adverse conditions but did not have any affect (p>0.05), positive or negative, on DNA integrity. A second phase of experiments was performed, narrowing the testing materials to a single additive, choline acetate, and increasing the sample size to improve statistical power. The addition of choline acetate in Phase 1 moderately improved recovery (p=0.055), while also improving handling procedures, observed as quick release and glass dissolution from the preservation material and facile collection. In Phase 2, the addition of choline acetate was demonstrated to statistically increase (p<0.05) GV recovery from substrates after exposure to adverse conditions when compared to trehalose alone and yielded equal recovery (p>0.05) under ideal conditions. Raman spectroscopy was used to confirm the state of the materials before and after humidity treatments. The control preservation composition of 20% weight/volume trehalose crystallized under adverse conditions, while the trehalose with the choline acetate additive 20% weight/volume remained amorphous. This study found that combining trehalose with a choline acetate salt additive enables good preservation outcomes even under transient high humidity conditions. This may enable more flexible transportation and storage of germinal vesicles that are to be used for reproduction purposes and potentially an overall reduction in the cost and complexity of genetic resource management.