MadSci Network: Cell Biology

Re: What is the function of egg yolk and glycerol in cryopreservation?

Date: Thu Jun 7 17:30:08 2001
Posted By: Elsa Lee, Grad student, Molecular Cell Biology, University of California, Berkeley
Area of science: Cell Biology
ID: 990002420.Cb

Dear Natalie,

I did a little research into your question.The following link has a detailed 
description regarding cryoprotectans used in equine sperm preservation 
Here's the short version:

There are two general classes of cryoprotectants: 1) penetrating 
cryoprotectants. These pass thorugh the sperm membrane and act both 
intracellularly and extracellularly, and 2) non-penetrating cryoprotectants, 
which act only extracellularly. 

Glycerol and DMSO are commonly ultilized penetrating cryoprotectants. 
Whereas non-penetrating cryoprotectants include proteins found in milk or  
egg yolk; sugars such as fructose , lactose, mannose, raffinose or 
trehalose; synthetic polymers such as polyvinylpyrrolidone or methyl 
cellulose and amides. Most penetrating cryoprotectants serve as both as a 
solvent and a solute. All non-penetrating cryoprotectants can not serve as a 

Glycerol serves as a solute within the water and also penetrates the sperm. 
Glycerol contributes to the osmotic pressure both outside and inside the 
spermatozoan. The non-penetrating cryoprotectants such as egg yolk, although 
are soluble, do not penetrate through the plasma membrane of a living cell 
Thus they contribute to the osmotic pressure of the extender solution. Non-
penetrating cryoprotectants move water out of the sperm which results in 
dehydration and shrinkage. When sperm is frozen for cyropreservation, the 
addition of glycerol helps prevent ice damage. Glycerol has a lower freezing 
temperature than water.

Apparently the process of thawing and insemination is potentially very 
damaging to the sperm due to rapid changes in volume.Frozen stocks
typically have an osmotic pressure >1000 mOsmol/kg whereas the environment 
of the uterus is >300 mOsmol/kg. This results in a rapid increase in volume 
for sperm as water moves in to equlibrate the high concentration of 
intracellular glycerol (left behind). This may result in rupture of the 
plasma membrane. 

There is also evidence that addition of egg yolk proteins to the freezing 
medium aids in the recovery of sperm motility.Here's the reference for that 
study: Alvarez JG, Storey BT. (1993) Evidence that membrane stress 
contributes more than lipid peroxidation to sublethal cryodamage in 
cryopreserved human sperm: glycerol and other polyols as sole 
cryoprotectant.J Androl May-Jun;14(3):199-209

Yildiz C, Kaya A, Aksoy M, Tekeli T. (2000) Influence of sugar 
supplementation of the extender on motility, viability and acrosomal 
integrity of dog spermatozoa during freezing.Theriogenology Sep 1;54(4):579-


To learn more about the osmotic effects of permeable versus non permeable 
reagents: Liu Z, Foote RH.(1998) Osmotic effects on volume and motility of 
bull sperm exposed to membrane permeable and nonpermeable agents.
Cryobiology 1998 Nov;37(3):207-18

Devireddy RV, Swanlund DJ, Roberts KP, Pryor JL, Bischof JC.(2000)
The effect of extracellular ice and cryoprotective agents on the water 
permeability parameters of human sperm plasma membrane during freezing.
Hum Reprod. May;15(5):1125-35.

Best wishes,



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