15 Jan 2015

Preserving genetic diversity

SASKATOON – Muhammad Anzar, a research scientist for Agriculture and Agri-Food Canada, is an expert in cryopreservation — you know, freezing living matter to cheat death. 

His work is more careful than comic book, with a focus not on preserving full organisms but the very germs of life: semen, oocytes and embryos.

“Basically my appointment in Agriculture Agri-Food Canada is about biodiversity and conservation. We are running a Canadian Animal Genetic Resource program to preserve the genetic diversity of our livestock species,” Anzar explains. 

In other words, his work is to protect rare livestock breeds from permanent disappearance by preserving their genetic material. In the case of bovine sperm, this is a fairly mundane task, but the female egg, or oocyte, is notoriously hard to preserve.

One of the main goals of cryopreservation is to cool the sample in question with a minimum of ice crystals, which are dangerous since freezing expands and tends to damage the cell’s internal workings. Ice damage varies species to species, animal to animal and even cell to cell, and the reasons for this variation have remained largely mysterious in the field. 

That’s why Anzar began work with Canadian Light Source CMCF beamline scientist Pawel Grochulski. CMCF is normally used to study protein structures from crystals, and both scientists thought the technique could be applied to finding troublesome ice crystals in the preserved oocytes.

It worked. For the first time, cryobiology had a way to observe the actual results of the cooling process in a cell. 

Now, ideally, instead of forming ice crystals in its cooled state, a properly cryopreserved cell will behave more like a glass — water and any preservatives used will form an amorphous state. The usual technique for this is called vitrification, literally to make a glass. 

In vitrification, most of the water in a cell is replaced with cryoprotectants and cooled ultra-rapidly.

“As soon as they are exposed to the high concentrations of cryoprotectants immediately they are plunged into liquid nitrogen with a temperature of about -196C,” said Anzar.

The cells cool at a rate of about 4000 to 5000 C a minute, so ice has almost no time to form, and the cell instead reaches a glass phase. 

Instead, CMCF analysis revealed that inside the bovine oocyte, ice crystals still continue to form. Cryoprotectant outside the cells formed glass, as expected, but something about the oocyte is resistant to preservation. In contrast, the bovine embryo was preserved in glass phase efficiently. 

Why this is true remains a mystery, but with this new ability to actually observe crystal formation in the vitrification process, Anzar has plenty of plans to continue the exploration.

“The next step might be to use fluorescent markers to tag things important for the cell health, and we’ll be able to study those biomarkers in the frozen state. Or we could look into what is preventing the cryoprotectant to go inside the oocyte.”

“This study opened a gateway to study the behavior of cells at low temperature, and that will be a new dimension for using CMCF to study the frozen cells.”

CLS CMCF Beamline Scientist Pawel Grolchuski with Agri-Food Canada and University of Saskatchewan scientist Muhammad Anzar.
Cite: Anzar, Muhammad, Pawel Grochulski, and Brennan Bonnet. "Synchrotron X-Ray Diffraction to Detect Glass or Ice Formation in the Vitrified Bovine Cumulus-Oocyte Complexes and Morulae." PloS one 9.12 (2014): e114801. DOI: 10.1371
This photo and others to accompany this story are available in the CLS image gallery for use with a Creative Commons licence.

About the CLS:

The Canadian Light Source is Canada’s national centre for synchrotron research and a global centre of excellence in synchrotron science and its applications. Located on the University of Saskatchewan campus in Saskatoon hosted over 2,000 researchers from academic institutions, government, and industry from 10 provinces and 2 territories; delivered over 32,000 experimental shifts; received over 8,300 user visits; and provided a scientific service critical in over 1,000 scientific publications, since beginning operations in 2005. The CLS has over 200 full-time employees.

CLS operations are funded by Canada Foundation for Innovation, Natural Sciences and Engineering Research Council, Western Economic Diversification Canada, National Research Council of Canada, Canadian Institutes of Health Research, the Government of Saskatchewan and the University of Saskatchewan.

Synchrotrons work by accelerating electrons in a tube to nearly the speed of light using powerful magnets and radio frequency waves. By manipulating the electrons, scientists can select different forms of very bright light using a spectrum of X-ray, infrared, and ultraviolet light to conduct experiments.

Synchrotrons are used to probe the structure of matter and analyze a host of physical, chemical, geological and biological processes. Information obtained by scientists can be used to help design new drugs, examine the structure of surfaces in order to develop more effective motor oils, build more powerful computer chips, develop new materials for safer medical implants, and help clean up mining wastes, to name a few applications.

For more information visit the CLS website or contact:

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mark.ferguson@lightsource.ca

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