X-Message-Number: 28756
From: "Basie" <>
Subject: Trehalose as cryo protectant
Date: Thu, 21 Dec 2006 13:56:31 -0500

Durable Critters Providing Insight For Human Egg Preservation
Science Daily - A tiny, six-legged critter that suspends all biological 
activity when the going gets tough may hold answers to a better way to 
cryopreserve human eggs, researchers say.


The durable tardigrade. (Credit: Rick Gillis and Roger J. Haro, Department 
of Biology, University of Wisconsin - La Crosse)Ads by Google Advertise on 
this site

Tardigrades, also called water bears, can survive Himalayan heights or ocean 
depths as long as they have moisture.

When they don't, they produce a sugar, trehalose, slowly dehydrate and 
essentially cease functioning until the rain comes, says Dr. Ali Eroglu, 
reproductive biologist and cryobiologist at the Medical College of Georgia.

Tardigrades are not alone in their amazing ability to outlast adverse 
conditions. A type of brine shrimp, often called a sea monkey, comes back to 
life with water. The Baker's yeast Dr. Eroglu uses when he bakes bread with 
his children does as well. Similarly arctic wood frogs use the sugar, 
glucose, to tolerate frigid temperatures until the summer thaw.

While humans don't naturally produce trehalose, researchers think they can 
use it to safely preserve human eggs -- and those of endangered species --  
giving better options to young women facing cancer therapies that may leave 
them infertile and others who simply want to delay reproduction.

"Our hypothesis is when we introduce sugars into cells and into oocytes, we 
can protect them against freezing-associated stresses," says Dr. Eroglu, who 
received a $1.2 million grant from the National Institute of Child Health 
and Human Development to continue pursuing his hypothesis. "We also 
hypothesized if we used trehalose, we also could use conventional 
cryoprotectants, which can be toxic, in lower concentrations to minimize 
their toxicity while maximizing overall protection."

Pilot data show it works like a charm, at least in mouse eggs. Researchers 
injected eggs with trehalose, cooled them to liquid nitrogen temperature, 
thawed them and exposed them to sperm. They got healthy babies at a similar 
rate to unfrozen controls.

"We were very excited," says Dr. Eroglu, whose work has prompted desperate 
calls from young cancer patients wanting to preserve their eggs. "We got 
very good development rates, then we transferred the embryos to foster 
mothers and got pups that were completely healthy." In fact, those pups had 
healthy pups. His limited testing in human eggs indicates they also can be 
preserved and thawed safely using this approach, however further research is 
needed to pursue clinical applications.

The NIH grant enables him to use monkey eggs, which are similar to human 
eggs, to find the optimal mix of sugar, conventional cryprotectants and 
freezing to maximize egg preservation. Collaborators at Emory University are 
providing the eggs and at the Georgia Institute of Technology are developing 
a mathematical model to predict cooling rates while avoiding destructive 
intracellular ice formation. Dr. Eroglu also is working with the MCG Section 
of Reproductive Endocrinology, Infertility and Genetics In vitro 
Fertilization Program to obtain discarded eggs that failed to fertilize.

Dr. Eroglu looks for a better way because current approaches are fraught 
with problems. Scientists have been freezing human eggs for about two 
decades but not very successfully. "Embryo cryopreservation is relatively 
successful, but to freeze oocytes, we have to overcome many hurdles," he 
says. A major problem is the protective, exterior jelly coat of an egg 
doesn't freeze well. The jelly coat protects the egg from mechanical stress 
and serves as a receptor for sperm. Sperm must pass through the coat then 
penetrate the interior plasma membrane. As soon as a single sperm 
penetrates, it triggers intracellular signaling that transforms the coat 
into a hard, impermeable structure and with good reason: if multiple sperm 
penetrate, chromosomal abnormalities result. Interestingly, traditional 
freezing, even with cryopreservatives, can cause these problems and more. 
The jelly coat hardens, making it impossible for sperm to get through the 
traditional way. "You don't have this issue with an embryo because 
fertilization already has occurred," Dr. Eroglu says.

Intracytoplasmic sperm injection came into use in 1997 to help overcome the 
hardened jelly coat but other problems persist. Chemical stress, freezing or 
warming can disrupt the egg's mechanism for dividing chromosomes -- babies 
get half their chromosomes from mom and half from dad -- so they don't line 
up as they should. In addition to hardening the jelly coat, cold stress can 
change intracellular signaling resulting from sperm penetration. Lipids or 
fats in the egg can fuse and the membrane can become leaky.

The bottom line is only about 1-5 percent of eggs develop to term after 
standard cryopreservation techniques, which include a combination of slow 
freezing in conjunction with low levels of cryoprotectants such as dimethyl 
sulfoxide, or rapid freezing with more cryoprotectants.

Dr. Eroglu says the sugars, which help protect the natural structure of 
proteins, enable use of warmer temperatures and fewer cryoprotectants. He 
uses the tried and true intracytoplasmic sperm injection approach to deliver 
sugar -- instead of sperm - to eggs before cooling. One of his ultimate 
goals is to design sugars that can easily penetrate the egg's membrane, but 
at least for now, tardigrade sugars seems to work just fine in mice.

One of his many hopes is that freezing embryos won't always be necessary, 
whether it's a human or an endangered species. Rather, eggs and sperm --  
which have been frozen successfully for decades -- can be kept apart until 
fertilization is desirable. This could preclude the controversy of 
destroying unused embryos and perhaps the debate over embryonic stem cells, 
he says.

Eggs, which can reprogram cell function by turning genes off and on, can 
produce cells that can become essentially anything, Dr. Eroglu says. If he 
can better understand how they do this, regular body cells might be 
reprogrammed in a test tube to embryonic-like stem cells for therapeutic 
use.

Note: This story has been adapted from a news release issued by Medical 
College of Georgia.

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