Different Effects of Sugars and Methods to Preserve Post-Thaw Functional Properties of Cryopreserved Caprine Spermatogonial Stem Cells

Abstract

The present study aimed to identify the effects of sugar and methods (slow freezing [SF] vs. fast freezing [FF]) on post-thaw in vitro functional characteristics of cryopreserved caprine spermatogonial stem cells (cSSCs) and the cells obtained from cryopreserved testis tissue of prepubertal Barbari bucks. For this, in experiment 1, cSSCs were isolated and cryopreserved by either SF or FF method with different non-permeable (sugars; trehalose [140 m<sc>m</sc>; 140T or 400 m<sc>m</sc>; 400T] and sucrose [140 m<sc>m</sc>; 140S or 400 m<sc>m</sc>; 400S]) or/and permeable (5% ethylene glycol [EG] and dimethyl sulfoxide) cryoprotectants. After 1 week of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment 2, the effectiveness of sugars (trehalose [140 m<sc>m</sc>] or sucrose [140 m<sc>m</sc>]) for cryopreservation of testicular tissues of prepubertal Barbari bucks using the SF or FF method was evaluated. After 1 week of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3 weeks. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers' expression. The recovery rate was 1.3-, 1.3-, and 1.1-fold higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (protein gene product 9.5 and octamer-binding transcription factor-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue weight was significantly (p &lt; 0.05) higher when cryopreserved using 140 m<sc>m</sc> trehalose compared with other groups. The results of immunocytochemical analyses imply the expression of pluripotent stem cell markers in cSSCs following cryopreservation. Overall, the outcome of the study demonstrates different effects of sugars and methods on post-thaw functional properties of cSSCs with superiority of 140 m<sc>m</sc> trehalose using SF method over other treatment groups. These results are important for ex vivo expansion and differentiation of cSSCs for fertility preservation and their other downstream applications.

Keywords: Caprine spermatogonial stem cells; Cryopreservation; Functional properties; Sugar; Testis.