Thawed spermatogonia

Rat sperm precursors remained functional after 23 years of freezing

American scientists have tested what happens to the precursors of rat germ cells during long storage in liquid nitrogen. It turned out that after defrosting and implantation into the testis, they are able to divide and produce spermatozoa. However, in this they still lose to both fresh progenitor cells and cells after a short freeze. This means that it can be difficult for people to use this strategy. The work was published in the journal plos Biology (Whelan et al., Reestablishment of spermatogenesis after more than 20 years of cryopreservation of rat spermatogonial stem cells reveals an important impact in differentiation capacity).

Freezing germ cells can be useful not only for those who want to prolong their reproductive period, but also for those who have not yet lived up to it. This problem is especially acute in children with oncological diagnoses. Doctors prescribe them therapy that can harm any rapidly dividing cells in the body, including sexual ones.

To give such patients the opportunity to have their own children when they cope with the disease and grow up, you can try to freeze their germ cells before therapy. But then these cells will have to spend at least years, or even decades, in a cryopreserved state. And it is still not entirely clear whether they can survive such a long period in freezing.

It is known that human embryos frozen in the early stages of development can survive frozen for at least 27 years. Human spermatozoa are at least 40 years old. But a small child does not have ready-made sperm yet, so their predecessors will have to be frozen. And how long they can last in liquid nitrogen and whether they will be functional after defrosting is still unknown.

A group of scientists led by Ralph L. Brinster from The University of Pennsylvania decided to test this on mice and rats. In their laboratory, spermatogonial stem cells (precursors of sperm cells) of rats, frozen 23 years ago, have been preserved. For comparison, they took similar cells frozen recently, 1-4 months ago, as well as fresh cells that were not frozen.

The researchers thawed cryopreserved cells and planted them in the testes of mice purified from their own cells (a special line with immunodeficiency, which does not have rejection). And they found that they all take root, but not all the same: after a long freeze, the progenitor cells were able to populate fewer tubules in the testes and produce fewer sperm cells than fresh cells or cells after a short freeze.

C is the number of colonies that produced transplanted cells in the testes, D is the proportion of tubules in which spermatozoa appeared. Figure from the article by Whelan et al.

Then the authors of the work decided to check whether these cells differ greatly in the work of genes. They measured gene expression in sperm progenitors immediately after defrosting and compared the results with unfrozen cells. It turned out that the duration of freezing does not greatly affect the work of genes, but fresh cells differ significantly from frozen ones.

However, when the researchers extracted cells that had already taken root in the testes of mice, they noticed that gene expression varies greatly, depending on how much the cells spent in liquid nitrogen. They noticed the same effect on the performance of these cells: those that had been frozen for 23 years produced fewer differentiated spermatozoa than recently frozen and fresh progenitor cells.

Thus, the authors of the work, on the one hand, checked that even 23 years in freezing do not deprive progenitor cells of the ability to take root inside the testis and produce germ cells. But, on the other hand, they found that after such a long shelf life, the cells cope with their duties worse. Therefore, researchers warn their colleagues working with human spermatozoa against spreading the results obtained on cells after a short freeze to cells after prolonged cryopreservation. And they are also encouraged to check not only the viability of cells after defrosting, but also their functionality — since problems with long-frozen cells arose after implantation into the testes.

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