Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats

Abstract

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Transcriptome dynamics in developing testes of domestic cats and impact of age on tissue resilience to cryopreservation

“…Mature testicular tissue from adult cats also was more responsive transcriptionally to vitrification compared to juvenile. Similarly, immature testes from domestic cat were reported to be more resilient to microwave-assisted dehydration [ 25 ] and more successful in surviving and establishing spermatogenesis in xenografts [ 51 , 52 ], compared to mature testes from adult cats. Specifically, we observed the downregulation of functional terms related to focal and cell-cell adhesion in adult compared to late juvenile, as well as to the whole juvenile group.…”

“…DNA integrity was assessed using the protocol reported previously for domestic cat testicular tissue using the In-Situ Cell Death Detection kit (Roche, Basel, Switzerland) [ 25 ]. Deparaffinized rehydrated sections were rinsed twice with 0.05 % Triton X-100 in PBS for 5 min each, permeabilized with 0.5 % Triton X-100 in PBS for 30 min, rinsed once with 0.05 % Triton X-100 in PBS for 5 min and incubated in TUNEL reaction mixture (enzyme solution with terminal deoxynucleotidyl transferase (TdT) and label solution with nucleotide polymers) for 1 h at 37 °C within a humidified darkened container.…”

“…Recently, our group observed a better recovery of immature testicular tissues compared to adult in domestic cat after preservation protocol using microwave-assisted drying [ 25 ]. Different separate studies also show that testicular tissue from immature cats tend to be more resilient to cryopreservation protocols compared to adults [ 26 – 28 ].…”

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

“…Present results were obtained in adult tissues, but it is expected that structural or functional properties in testes from prepubertal or younger BFFs would be comparable or even better, as seen in the preservation of cat testicular tissues [15]. Collective results helped to better characterize the resilience of BFF testicular tissues to freezing temperatures and the essential role of rapid warming followed by a 24-h reanimation period.…”

“…Previous studies reported that germ cells at the early stages of spermatogenesis are more sensitive to DNA damage than sperm cells. Non-physiological conditions created by the preservation methods break hydrogen bonds between the bases of the DNA strands [15]. 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].…”