Influence of warming and reanimation conditions on seminiferous tubule morphology, mitochondrial activity, and cell composition of vitrified testicular tissues in the domestic cat model

Abstract

Understanding critical roles of warming and reanimation is critical to improve the survival of vitrified testicular tissue in domestic cats. The objective was to study structural and functional properties of testicular tissues from prepubertal domestic cats after standard vitrification followed by two warming protocols (directly at 37°C or with a 5-second pre-exposure to 50°C) and three reanimation time points (immediately, 24 h and 5 days post-warming). In Experiment 1, tissues were evaluated for histo-morphology and mitochondrial activity immediately or 24 h after warming protocols. In Experiment 2, cell viability, DNA fragmentation, and germ cell composition were assessed immediately, 24 h, or 5 days after optimal warming. Preservation of seminiferous tubule structure was better using warming at 50°C for five seconds, and survival of somatic as well as germinal cells was higher compared to direct warming at 37°C for one minute. Short term in vitro culture (for reanimation) also proved that cellular composition and functionality were better preserved when warmed for a short time at 50°C. Collective data showed that short warming at 50°C led to better quality of seminiferous tubule structure and cell composition after vitrification and short-term culture. In addition, data suggest clear directions to further understand and optimize testicular tissue survival after fertility preservation procedures.

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

“…Tissue biopsies were immediately evaluated (fresh control; see Experimental Design) or exposed to a mix of cryoprotectants (dimethylsulphoxide (DMSO) and glycerol (Sigma- Aldrich) following our standard protocol [ 7 ]. Briefly, tissue biopsies were threaded onto a 30-G needle (BD Precision Glide needle, Fischer Scientific, Waltham, MA, USA) and immersed in an equilibrium (1.4 M of each cryoprotectant plus 0.25 M sucrose in Ham’s F10) for 10 min at room temperature (~22–24 °C).…”

“…Tissues were fixed overnight in Bouin’s solution before embedding in paraffin and serial sections (5-µm thick). After mounting sections on frosted glass slides, staining with hematoxylin-eosin was performed according to a standard protocol [ 7 ]. Slides then were observed using an upright microscope fitted with digital photomicrography (SPOT advanced software 5.0; Diagnostic Instruments, Sterling Heights, MI, USA).…”

“…Seminiferous tubule and cell integrity were evaluated based on the attachment of cells to the basal membrane, absence of breaks in the stroma, no swelling of the lamina propria, and tight junctions between cells. Structures received a score of 1 in that case [ 7 ]. If any change was observed in the previous criteria, tubules received a score of 0.…”

“…Proper warming can prevent devitrification/ice recrystallization and ensure optimal reanimation in preserved tissues. Recently, beneficial effects of short time exposure to high warming temperatures have been reported in testicular tissues from prepubertal cats [ 7 ]. An additional short culture period of warmed tissues also promotes cell survival and mitigates DNA injuries [ 7 , 8 ].…”

“…Sertoli cells are critical to fully support germ cell development and differentiation throughout spermatogenesis [ 11 ]. Thus, the structure and functions of seminiferous tubules, including connections between germ cells (histomorphometry) and the integrity of Sertoli cells (assessed by the presence of vimentin), have to be properly preserved [ 6 , 7 ]. Recent studies in the cat model confirmed that markers such as Oct4 and Boule are present in pre-meiotic (spermatogonia) and meiotic (spermatocytes) germ cells, respectively [ 7 ].…”