ROS Are Generated by Osmotic Stress During Cryopreservation

Several steps in a typical cryopreservation protocol are responsible for the osmotic stress that cells experience. First, during the loading of cells with cryopreservative agent (CPA), they typically experience a hyperosmotic stress and shrink on initial exposure to 1 M glycerol. This initial and immediate response is linear (an ideal osmometer) and occurs by passive membrane diffusion in which water travels down its concentration gradient and requires no cellular control. This adjustment in cell volume follows the Boyle van't Hoff (BVH) relationship,

MiS0

1 -

V,

+ Vl

M

Vis _

Vis

The BVH equation for determination of cell volume: Vb is the inactive cell volume that is excluded from osmotic response [11].

and is known to occur in sperm in usually less than 1-2 s. Subsequently, the cells re-expand as they equilibrate to the CPA. Then, if volume regulation occurs, there will be a slow change in volume over a time period of minutes to tens of minutes. This final change would be evidence of regulated volume increase or decrease (RVI or RVD). They return to near-isotonic volume (but slightly expanded because of the CPA loading). Next, the cells are exposed to a slow cooling protocol. During this phase of cryopreservation, the cells are dehydrated and typically must lose about 90% of their cell water to survive [11, 93] which means substantial shrinkage of the cell: the exact amount depending on the inactive cell volume, Vb. Finally, after thawing, cryoprotectant must be washed out of the cell. If done in a single step, the cell, which is now internally in a hyperosmotic state because of the presence of CPA, is placed in an isotonic solution. The cell responds by rapidly swelling in volume followed by a slower return to isosmotic volume as the CPA leaves the cell. Although most spermatozoa have narrowly defined osmolal conditions in which cell function is lost, a mechanism for this loss in cell function has not been determined. Since sperm cells emerge from freezing with primarily loss of function (motility) rather than compromised structure, it is likely that events other than formation of intracellular ice contribute to cryodam-age of these cells.

Most cells, including spermatozoa, are capable of regulating their intracellular osmotic balance and, hence, cell volume, when exposed to osmotic challenge. This is a highly conserved cell function and is extremely important for homeo-static balance in mammals for essentially all cell functions. This volume regulation is linked to function of the cell's cytoskeleton and plasma membrane system. Spermatozoa are exposed to various environments during their life in vivo that impart osmotic challenges. The distal cauda epididymis, for example, is known to be significantly hypertonic in comparison to testicular and caput epididymal fluids. Consequently, when sperm are ejaculated, they encounter a rapid exposure to the relative hypotonicity of the seminal fluid and female reproductive tract, and swell by osmosis. However, excessive cell volume change, as when exposed to cryopreservation, could impair cellular regulatory and metabolic components, resulting in detrimental changes including cell lysing. Volume regulation is important to sperm function and may be damaged by cryopreservation as phos-pholipid membranes are physically stretched during the process resulting in plasma membrane and mitochondrial injury. When sperm are exposed to anisosmotic conditions, the volume change has been associated with increases in phosphorylation of protein tyrosine residues, a signaling event coincidentally required for sperm capacitation and fertilization [16, 36, 88, 94-96]. It has been hypothesized that when sperm undergo slow freezing, the ability to volume regulate, recover motility, and respond to subsequent post-insemination environments may be compromised. Therefore, the result could be decreased or compromised fertility.

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