Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation

Authors: Purdy, Phil; Spiller, Scott; Blackburn, Harvey

Submitted to: Cryobiology
Publication Type: Abstract Only
Publication Acceptance Date: 6/30/2013
Publication Date: 12/13/2013
Citation: Purdy, P.H., Spiller, S.F., Blackburn, H.D. 2013. Rooster sperm plasma membrane protein and phospholipid organization and reorganization attributed to cooling and cryopreservation. Cryobiology. https://doi.org/10.1016/j.cryobiol.2013.09.035.
DOI: https://doi.org/10.1016/j.cryobiol.2013.09.035

Interpretive Summary: Frequently, animal cell membrane pliability is used to assess the potential for a cell to survive freezing and predict the post-thaw quality following storage but the results of this assessment are not consistent across species and only evaluate the effects of one component, lipids while minimizing the potential impact of another component, proteins. Therefore, this research explored the interactions of these components during the freezing process of rooster sperm in order to potentially enable identification of poor samples prior to freezing. Our results demonstrate that when analyzed separately, they can be used to predict post-thaw quality and when combined a more accurate prediction of post-thaw quality can be generated. Consequently, these results provide scientists and commercial entities that freeze semen for artificial insemination with a better understanding of a membrane during the freezing process. This may, after further exploration, be used as an assay that can identify inferior samples prior to freezing and thus save valuable time and resources by only freezing samples deemed to be of a higher quality.

Technical Abstract: A plasma membrane that can respond to physical and chemical stresses is requisite in order for a cell to survive cryopreservation. The dynamic nature of a membrane is determined by the components (cholesterol, phospholipids, protein) which, in conjunction with environmental factors such as temperature and diluent, determine the plasma membrane organization and consequential fluidity. Frequently, the membrane fluidity, defined as the cholesterol to phospholipid ratio, is used to assess the potential for cryopreservation success and predict the post-thaw quality. Still, the results of this assessment are not consistent across species and only evaluate the effects of cholesterol and phospholipids while minimizing the potential impact of membrane proteins. For that reason, this research explored the interactions of membrane fluidity and protein organization during the cooling and cryopreservation process of rooster sperm using the fluorescent stains merocyanine 540 and rhodamine 640 to assess plasma membrane phospholipid and protein organization, respectively. Our results demonstrate that when analyzed separately, membrane phospholipid and protein organization can be used to predict post-thaw quality. What's more, an interaction of phospholipid and protein reorganization can be modeled during the cryopreservation process when the results of both analyses are combined and can be used to predict the post-thaw quality. This reorganization is dependent upon the cryopreservation diluent, penetrating cryoprotectant, and environmental temperature. Consequently, these results demonstrate the kinetic nature of the rooster sperm plasma membrane and present an assay that, following additional investigation, may potentially be used to monitor the quality of sperm samples.