Spermatogonial stem cells (SSCs) are the baseline cells of spermatogenesis. They have gained a lot of attention during the last few decades due to their great potential in biotechnology. Namely, as stem cells, SSCs can either self-renew or commit to differentiation through which they produce male gametes – spermatozoa. Okutsu et al. (2007) were the first to observe that when transplanted into recipients of a closely-related species, these cells incorporate into recipient’s genital ridge, proliferate, and differentiate into donor-derived gametes ultimately giving donor-derived progeny. This commenced the research on a novel reproductive biotechnology known as the surrogate production technology.
The surrogate production technology entails the following steps: isolation of SSCs from the donor individuals, sterilization of recipient larvae, transplantation of SSCs into recipient larvae and rearing of recipients. The success of the method depends on the availability of donors and recipients of the appropriate life stage and the availability of good quality germ cells from the donor. To help synchronize the procedure, short- or long-term storage are necessary. The most used methodology for SSC storage is cryopreservation which enables their storage for theoretically indefinite time. SSC cryopreservation protocols have been developed for many fish species, and the aim of this study is to summarize the main conclusions of these studies.
SSCs can be frozen either within the whole testicular tissue, or as isolated cells within the cell suspension. Usually, they are cryopreserved within the testicular tissue, and Pšenička et al. (2016) and Marinović et al. (2017) displayed that freezing within tissues yielded higher viability. The tissue can then be either frozen through slow-rate freezing or vitrified. The choice of the approach should mostly depend on the tissue structure, i.e., whether the testis is mature and contains a lot of spermatozoa, or if it is immature and contains only early-stage germ cells. Most commonly, when the tissue is mature and contains a lot of spermatozoa, freezing is a much better option as vitrification gives very poor results. This is consistent with the poor performance of vitrification for cryopreserving spermatozoa in fish. Next, the cryomedium should be optimized. Both freezing and vitrification outcomes mostly depend on the type and concentration of the permeable cryoprotectant, while non-permeable cryoprotectants (sugars, BSA, FBS) affect the outcome to a much lesser extent. In most of the studies, dimethyl sulfoxide and ethylene glycol yield the most favorable SSC viability after freezing. During vitrification, much higher cryoprotectant concentrations are needed, therefore using equal amounts of two different cryoprotectants yields better result than a very high concentration of a single cryoprotectant. The cooling rate during freezing is mostly kept around -1 ℃/min, and several studies that have tested various rates have confirmed the superior performance of this rate.
Cryopreservation of SSCs is a very valuable method, however, without further application, the benefit of cryopreservation is only limited to creating gene banks and frozen zoos. Therefore, development of the surrogate production technology of cell culture technology is essential for the utilization of the cryopreserved SSCs.
Acknowledgement
The work/publication is supported by the National Research, Development and Innovation Office of Hungary (projects K138425 and FK142933).
References
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Okutsu T, Shikina S, Kanno M, Takeuchi Y, Yoshizaki G. Production of trout offspring from triploid salmon parents. Science (80- ) 2007;317:1517. https://doi.org/10.1126/science.1145626.
Pšenička M, Saito T, Rodina M, Dzyuba B. Cryopreservation of early stage Siberian sturgeon Acipenser baerii germ cells, comparison of whole tissue and dissociated cells. Cryobiology 2016;72:119–22. https://doi.org/10.1016/j.cryobiol.2016.02.005.