APPLICATION OF BIOPOLYMERS FOR CRYOPRESERVATION OF RAT TESTICULAR TISSUE

Abstract

Today, transplantation of cryopreserved fragments of immature testicles is a non-alternative way for fertility preserving in pre-adolescent patients, which has been planed cytotoxic therapy. However, loss of spermatogonal stem cells occurs during cryopreservation. Therefore, the effectiveness of the cryopreservation procedure is critical and should be improved. A promising approach is to use biopolymer gels, since the presence of an extracellular matrix may affect the structure of ice crystals during cryopreservation. The aim of the work was to determine the effect of collagen (CG) and fibrin (FG) gels on the morphological and functional characteristics of the fragments of the seminiferous tubules of the testes of immature rats in a programmed freezing condition. Object and methods. The following experimental cryoprotective media were used: 1. collagen gel (CG) with 5% DMSO; 2. fibrin gel (FG) with 5% DMSO. Controls: 1. Hanks’ solution with 5% bovine serum albumin (BSA) and 5% DMSO; 2. Hanks’ solution without cryoprotectant; 3. intact tissue. CG was prepared from collagen type I, which was obtained from rat tendons. FG was obtained from an average fraction of fresh blood of animals after centrifugation (12 min, 1500 g). Samples of seminiferous tubules were obtained mechanically from both testes of immature rats (n = 50, weighing 50 ± 15 g, aged 7-8 weeks), incubated in media for 30 min (4°C) and cryopreserved according to the program: ramp to 0°C at a rate of 1°C/min; hold for 5 min at 0°Ñ; ramp to -8°Ñ at a rate of 1°Ñ/min; hold for 1 min at -8°C; ramp to -40°C at a rate of 1°C/min and up to -70°C at a rate of 10°C/min; transferred to liquid nitrogen. After heating the histological structure and the metabolic activity (MTT test and LDH activity) of spermatogenic epithelium cells was evaluated. Results. Histologically, testicular tissue of intact rats had a normal structural organization. Cryopreservation without cryoprotectants (negative control) caused gross damages of structure of seminiferous tubules: a sharp retraction of cells with the formation of large cracks inside the spermatogenic epithelium, its complete desquamation, lysis and picnosis of almost 90% of nuclei. The spermatogenic epithelium after cryopreservation under protection of BSA + 5% DMSO had moderately pronounced changes: the degree of retraction and desquamation, the number and size of the cavities decreased. The use of FG with 5% DMSO (as opposed to CG) led to a decrease in the severity of desquamation and retraction of spermatogenic cells, as well as in the number of cells with pyknotic nuclei, compared to the use of BSA instead biopolymer gel. According to the MTT-test, cryopreservation under the protection of FG with 5% DMSO was in 3.9 times more effective compaid to the negative control, exceeding by 27% the result of application of BSA as the basis of the cryoprotective medium. A similar trend was observed during LDH activity determination: in the group of FG + 5% DMSO application LDG activity was increased in 1.6 times in relation to negative control. The use of CG was less effective by both parameters. In general, the obtained data indicate that the use of FG as a basis of cryoprotective medium increases the resistance of the cells of the seminiferous tubules of immature rats to the action of factors of cryopreservation. Conclusion. Cryopreservation of fragments of the seminiferous tubules under the protection of a cryoprotective medium based on FG allows preserving their histostructure and metabolic activity and is more effective than the use of BSA or CG.

Keywords

• testicular tissue
• immature rats
• dimethyl sulfoxide
• cryopreservation
• bovine serum albumin
• collagen gel
• fibrin gel.