Cryoprotective agents (CPAs) are frequently used as part of a cryopreservation protocol. When added to a sample before freezing, CPAs can: 1) reduce damage to cellular components from intracellular ice formation and 2) promote recovery upon thawing.
But, determining the best type and concentration of CPA to use isn’t always easy. Too little, and there’s no benefit; too much, and you could destroy the very cells you are trying to preserve.
The cellular pathways involved in CPA-assisted cryopreservation are currently unknown, and biobankers typically scour the literature to pin down the optimal CPA type and concentration for their samples. That’s why I found a recent paper from Cryobiology particularly interesting.
In Effects of glycerol on apoptotic signaling pathways during boar spermatozoa cryopreservation, Changjun Zeng and colleagues examine the effect of glycerol CPA concentration on post-thaw recovery of boar sperm. The authors drill down deeper than simply providing recovery numbers at different concentrations, though. They actually try to explain the mechanism of glycerol as a CPA.
What do Zeng et al. propose?
Well, to better understand how glycerol effects sperm recovery, the authors measured the expression of various genes related to apoptosis under different concentrations of the CPA.
Apoptosis, or programmed cell death, is an important part of cellular regulation in mammals and other organisms. Apoptosis and related pathways can be activated in response to cellular damage and stress from external sources. Unfortunately, cryopreservation and thawing is stressful enough to activate this pathway in some cells, reducing post-thaw recovery rates.
The authors found that a key marker of apoptosis was lowest after thawing when cells were preserved in 2 to 3% glycerol. In addition, markers for “anti-apoptotic effects’’ were highest at these same concentrations.
The gene expression analysis also correlated well with traditional measures of sperm preservation. Both the viability and motility of sperm post-thaw were highest when preserved with 3% glycerol vs. other glycerol concentrations and a control sample without CPA. The authors conclude that 2-3% glycerol is the best concentration range for boar sperm with respect to motility post-thaw and gene expression patterns.
Even so, the relationship between glycerol and apoptosis is not 100% clear. Other markers for apoptosis actually increased with the presence of glycerol, indicating the CPA still could be damaging to the sperm cells in some way and that further research is needed to fully understand the cellular mechanisms involved when using CPA to enhance cryopreservation.
Studies like these are important because they can help develop better cryopreservation protocols. Indeed, glycerol is often used in the preservation of other sperm samples, as well. Biobanks like the Frozen Zoo depend on cryopreserved sperm and artificial insemination to preserve animal diversity in the event of future population declines or extinctions.
What’s more, this research underscores the need to tune cryopreservation protocols to the specific type of sample being preserved. 2 or 3% glycerol may be optimal for boar sperm, but could be detrimental to a different cell type or sperm from other animals. As I’ve said in the past, cryopreservation is definitely not a “one size fits all” process!