Cryobiology is the study of life at subzero temperatures. But our interest in it is other than creating new lifestyles for survival in glacial environments. Proteins, cells, small pieces of tissues, or whole organs can be stored at very low temperatures, which allows us to take advantage, in the future, of sources we have available today.
An example of the applicability of this technique is the cryopreservation of germ cells (sperm cells and oocytes) or embryos until a suitable individual is available for providing the miracle of life to happen. Now, our ambition is the possibility of bringing to life pups of endangered species while working with cryopreservation of domestic cat ovarian tissue.
Cats show phylogenetic and physiological similarities to felids that are under risk of extinction; in addition, their reproductive tissue is widely available after spaying is performed in veterinary clinics. We used ovaries of domestic cats with the aim of developing a tailored method for cryopreserving this type of tissue. By developing this technique, we will be able to adapt it for use in ovaries of wild felids that died or need to be spayed. After thawing, the germ cells can be reactivated in vitro or even in vivo, through the promising technique of ovarian tissue transplantation.
One of the obstacles faced in cryobiology is the formation of ice inside the tissue, detailed in Figure 1. To avoid that, a solution containing a cryoprotectant compound is usually applied promoting the externalization of water from the cells. We tested three different solutions containing DMSO, ethylene glycol, or a combination of both in order to see which allows a better maintenance of cell structure after the tissue is thawed. An adapted pattern of temperature reduction was performed and the samples were stored at -196°C in liquid nitrogen.
Figure 1. Animal tissues consist in an aqueous environment. In ordinary conditions, when temperatures decrease to subzero levels, the water molecules tend to rearrange and crystalize, causing physical damages to the cells. To avoid this effect, cryopreservation protocols are tested using cryoprotectant solutions that tend to remove water from the tissue, avoiding excessive crystallization. Credit: Ellen Leonel
After thawing, we analyzed the tissue by light and also transmission electron microscopy, which enables analysis at very high magnifications, allowing the evaluation of subcellular compounds.
We found out that the solution containing DMSO in a concentration of 1.5 M was the best in terms of preserving the oocytes’ morphology. The other solutions caused injuries, such as detachment between neighboring cells, the formation of vesicles in the cytoplasm, and disruption of the plasmatic membrane and nuclear envelope. Such damages would not allow cells to reestablish their activity after thawing, probably leading to death.
Next, we aim to find out if, in addition, to maintain the cell structure, the cryopreservation protocol was able to maintain the oocyte feasibility. To induce the development of germ cells present in the frozen ovarian tissue either, in vitro culture or transplantation techniques may be applied. Our next step is to perform these tests in order to conclude that the cryopreserved oocytes have the capacity to grow and develop in order to be fertilized.
These findings lead us to conclude that we are on the right track toward cryopreservation of wild felids’ ovarian tissue. Meanwhile, there is still a lot to be done. We are now studying ways to improve both the cryopreservation and the transplantations techniques and, in the future, be able to develop frozen-thawed oocytes of one felid species into another one.
These findings are described in the article entitled Cat Ovarian Follicle Ultrastructure after Cryopreservation with Ethylene Glycol and Dimethyl Sulfoxide, recently published in the journal Cryobiology. This work was conducted by Ellen C. R. Leonel, Janice M. V. Vilela, Daniela J. Carrilho and Carolina M. Lucci from University of Brasília, Brazil.