Cryopreservation and Culture of Testicular Tissues: An Essential Tool for Biodiversity Preservation

Abstract

Systematic cryo-banking of reproductive tissues could enhance reproductive management and ensure sustainability of rare mammalian genotypes. Testicular tissues contain a vast number of germ cells, including at early stages (spermatogonia and spermatocytes), that can potentially develop into viable spermatozoa after grafting or culture in vitro, and the resulting sperm cells then can be used for assisted reproductive techniques. The objective of this review was to describe current advances, limitations, and perspectives related to the use of testicular tissue preservation as a strategy for the conservation of male fertility. Testes can be obtained from mature or prepubertal individuals, immediately postmortem or by orchiectomy, but testicular biopsies could also be an alternative to collect samples from living individuals. Testicular fragments can be then cryopreserved by using slow or ultra-rapid freezing, or even vitrification methods. The composition of cryopreservation media can vary according to species-specific characteristics, especially regarding the cryoprotectant type and concentration. Finally, spermatozoa have been usually obtained after xenografting of testicular fragments into severely immunodeficient mice, while this method still has to be optimized after in vitro culture conditions.

Long-Term Preservation of Testicular Tissue Integrity and Viability Using Vitrification in the Endangered Black-Footed Ferret (Mustela nigripes)

“…We hypothesize that more cells had time to revive over 24 h of culture and eventually were present in higher proportions than in the refrigerated/fresh tissue shipped overnight to our laboratory. This is a phenomenon that has been already reported in gonadal tissues in many species (from ungulates to carnivores) following vitrification and warming [ 3 , 18 ].…”

“…Our next step is to decipher and quantify DNA repair mechanisms in tissues through staining of proliferating cell nuclear antigen (PCNA) or RAD51 protein [ 16 ] and detection of heat shock proteins [ 17 ]. Similarly, testicular cell viability improved after culture like in other species, which is considered a good sign of functional reanimations [ 3 ]. Interestingly, viability after vitrification and culture was even better than in fresh tissues.…”

“…Therefore, it is critical to collect or rescue and store genetic materials from all individuals for later use. As seen in other rare and endangered species, long-term preservation of ovarian and testicular tissues from individuals sterilized for medical reasons or dying unexpectedly offers additional options to study and propagate genetically valuable animals over multiple generations [ 3 ]. Cryopreservation of testicular tissues is now conducted in humans, laboratory models, domestic species, and some wildlife [ 3 , 4 , 5 ].…”

“…Among the multiple preservation protocols that are available for testicular tissues, vitrification or ultra-rapid freezing is the most convenient approach that has already provided encouraging results for carnivore tissues regarding the maintenance of histomorphometry [ 3 , 6 ]. While cryoprotectants, exposure conditions, tissue biopsy size, or freezing rates play critical roles in tissue survival [ 3 ], warming is an essential step that is often overlooked. Proper warming can prevent devitrification/ice recrystallization and ensure optimal reanimation in preserved tissues.…”

“…Interestingly, spermatozoa from frozen-thawed testicular tissues are viable in the cat model (based on the birth of healthy offspring after transfer of embryos produced by sperm injection) [ 9 ]. However, testicular tissues contain a large number of germ cells at early stages (in seminiferous tubules) that have the potential to develop into mature sperm cells after in vitro culture or xenografting as seen in other species, including the domestic ferret [ 3 , 10 ]. Spermatogenesis requires adequate germ cell support that may be compromised during freezing/warming as seen in humans [ 5 ] or in the cat model [ 6 ].…”