EFFECT OF CRYOPROTECTANT EQUILIBRATION ON TRANSMEMBRANE POTENTIAL OF BOVINE OOCYTES

A major physiological event during cryopreservation is equilibration to and from high concentrations of cryoprotective agents. The focus of this study was to analyze the effect of large and rapid osmotically induced volume perturbation on the transmembrane potential of bovine oocytes. The aim of this study was to evaluate and examine the difference in putative effects of non-permeating (PBS) and permeating (10% EG) solutions and to explore the association between osmotically induced volume changes on voltage in bovine oocytes. Oocytes were exposed to alternating sequences of PBS treatments i.e., initial hypotonic followed by hypertonic (from isosmotic to 0.5x or 0.3x, followed by isosmotic and then to 2x) and initial hypertonic followed by hypotonic (isosmotic to 2x, followed by isosmotic again and then to 0.3x or 0.5x). For isosmotic (iso) exposure, 1x PBS was used, for hypotonic, 0.3x or 0.5x of PBS was used whereas for hypertonic concentrations, 2x PBS was used. For the permeating solution, oocytes were exposed to 10% ethylene glycol. The change in the membrane potential was recorded using an intracellular electrode and the cell volume was measured using ImageJ software. The baseline resting membrane potential was recorded before the start of experiment and the voltage change was recorded during exposure to sequences of anisosmotic conditions. The membrane voltage hyperpolarized in the hypotonic solution and depolarized in a hypertonic solution. The relationship of normalized volume vs voltage showed negative slopes of -9.9 mV (R2 = 0.36) and - 49.65 mV (R2 = 0.56) in permeating and non-permeating treatments respectively which indicated a negative correlation between volume vs voltage. Welch’s t-test indicated a significant difference between slopes of volume vs voltage in permeating and non-permeating groups (t = 2.58 and p = 0.02). Overall, the exposure to anisosmotic PBS and 10 % EG caused differential change of voltage in bovine oocytes. The change in voltage was biphasic in permeating group as compared with non-permeating group. Within non-permeating treatment categories, there was a significant difference between iso to 0.3x PBS before pretreatment with hypertonic solution and iso to 0.5x PBS after pretreatment with hypertonic solution. Overall, the osmotically induced volume change has more impact on transmembrane potential in the presence of non-permeating solutions versus a permeating solution. Additionally, there was also a significant difference in the volume-voltage slope between two non-permeating sub-groups.

Name thesis:degree_name

Master of Science (M.Sc.)

Level thesis:degree_level

Masters

Discipline thesis:degree_discipline

Biology

Grantor

University of Saskatchewan

Year dc:date.issued

2024

Author dc:creator

• Hamza, Faryal

Advisors dc:contributor.advisor

• Benson, James
• Gray, Jack

Committee members dc:contributor.committeemember

• Niyogi, Som
• Howland, John

• Bovine oocyte cryopreservation, Transmembrane potential, intracellular recording from bovine oocytes, cryoprotectants induced voltage changes in oocytes, volume vs voltage relation in oocytes

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/10388/15439

OAI identifier oai:identifier

oai:harvest.usask.ca:10388/15439