Efficiency of Combined Use of Fullerene C60 and Bovine Serum Albumin for Rehabilitation of Vitrified Fragments of Rat Immature Seminiferous Tubules





fullerene C60, bovine serum albumin, seminiferous tubules, vitrification


Background. Today, cryopreserved reproductive tissues are used to treat some forms of male infertility. However, after long-term preservation of fragments of seminiferous tubules of testes (FSTT) in a low-temperature bank (-196 °С) their morphological and functional characteristics decrease reversibly. To solve this problem after freezing-thawing, the use of rehabilitation media with special additives is promising. Due to the fact that serum albumin and fullerene C60 have powerful protective and antioxidant properties, their use allows to stabilize the plasma membrane, osmotic pressure, and reduce free radicals that make them promising candidates to use in the development of rehabilitation media for biological objects after cryopreservation.

Objective. The efficacy of fullerene C60, bovine serum albumin (BSA), and their combination as components of rehabilitation medium of vitrified FSTT of immature rats was studied.

Methods. Vitrified-thawed samples of FSTT were incubated (22 °C) for 30 minutes in Leibovitz's medium with addition of 15 mg/mL C60, 5 g/L BSA or their combination. Control samples were incubated in the medium without C60 or BSA addition. Metabolic activity (MTT test), histomorphological data, total antioxidant status (TAS), reactive oxygen species (ROS) production, activity of g-glutamyltransferase (gGGT), and glucose-6-phosphate dehydrogenase were determined in the samples after rehabilitation in the investigated media.

Results. The use of C60 led to the increase of metabolic (by 1.26 times) and TAS (by 1.74 times) activities, to the decrease in the number of ROS+ cells (by 1.35 times) and to the improvement of the spermatogenic epithelium binding to the basement membrane versus control sample. Application of BSA did not significantly affect the studied biochemical indices but decreased the number of tubules with desquamation of spermatogenic epithelium in histological sections. The combined use of BSA and C60 had the best effect among investigated rehabilitation media that led to the increase of metabolic activity (by 1.51 times), TAS activity (by 1.78 times), gGGT activity (by 1.59 times), histostructure restoration and the decrease in the number of ROS+ cells (by 1.45 times) compared to the control samples.

Conclusions. The use of C60 and BSA combination increases the metabolic and antioxidant activity of vitrified FSTT and also has a positive effect on their histostructural characteristics compared to control samples. It should be noted that the effect of С60 and BSA addition to rehabilitation medium exceeds the results of using the investigated additives separately (by the metabolic and gGGT activity as well as architectonics of vitrified FSTT). These data relate to reproductive medicine and can be used to develop an effective rehabilitation protocol for vitrified FSTT.


Song Y, Sharp R, Lu F, Hassan M. The future potential of cryopreservation for assisted reproduction. Cryobiology. 2010;60(3):S60-5. DOI: 10.1016/j.cryobiol.2009.09.009

Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408(6809):239-47. DOI: 10.1038/35041687

De Leeuw FE, De Leeuw AM, Den Daas JH, Colenbrander B, Verkleij AJ. Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing. Cryobiology. 1993;30(1):32-44. DOI: 10.1006/cryo.1993.1005

Dobretsov GYe. Fluorescent probes in the study of cells, membranes and lipoproteins. Moscow: Nauka; 1989. 277 p.

Volkova NO, Yukhta MS, Chernyshenko LG, Stepanyuk LV, Sokol LV, Goltsev AM. Exposure of seminiferous tubules of immature rats to cryoprotective media of various compositions. Problems Cryobiol Cryomed. 2017;27(3):203-18. DOI: 10.15407/cryo27.03.203

Ritter U, Prylutskyy YI, Evstigneev MP, Davidenko NA, Cherepanov VV, Senenko AI, et al. Structural features of highly stable reproducible C 60 fullerene aqueous colloid solution probed by various techniques. Fuller Nanotub Carbon Nanostruct. 2014;23(6):530-4. DOI: 10.1080/1536383X.2013.870900

Fazylov SD, Nurkenov ОА, Muldakhmetov ZM, Gazaliev AM, Аrinova АЕ, Ibraev MK, et al. Biologically active deriva-tives of fullerene C60. Current state and development prospects. Bulletin of the Karaganda University. 2020;99:11-20. DOI: 10.31489/2020Ch3/11-20

Emelyantsev S, Prazdnova E, Chistyakov V, Alperovich I. Biological effects of C60 fullerene revealed with bacterial biosensor-toxic or rather antioxidant? Biosensors (Basel). 2019;9(2):81. DOI: 10.3390/bios9020081

Volkova N, Yukhta M, Goltsev A. Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats. Cell Tissue Bank. 2018;19(4):819-26. DOI: 10.1007/s10561-018-9740-z

Volkova NO, Yukhta MS, Goltsev AM. Vitrification of rat testicular tissue using biopolymers. Biopolym Cell. 2020;36(2):122-32. DOI: 10.7124/bc.000A26

Volkova N, Yukhta M, Sokil L, Chernyshenko L, Stepaniuk L, Goltsev A. Determination of warming modes for seminiferous tubules of rat’s testes after vitrification. Problems Cryobiol Cryomed. 2021;31(1):95-9. DOI: 10.15407/cryo31.01.095

Scharff P, Ritter U, Matyshevska OP, Prylutska SV, Grynyuk II, Golub AA, et al. Therapeutic reactive oxygen generation. Tumori. 2008;94(2):278-83.

Siu MK, Cheng CY. Extracellular matrix: recent advances on its role in junction dynamics in the seminiferous epithelium during spermatogenesis. Biol Reprod. 2004;71(2):375-91. DOI: 10.1095/biolreprod.104.028225

Bakry R, Vallant RM, Najam-ul-Haq M, Rainer M, Szabo Z, Huck CW, et al. Medicinal applications of fullerenes. Int J Nanomed. 2007;2(4):639-49.

Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl. 2008;29(5):488-98. DOI: 10.2164/jandrol.108.005132

Domínguez-Vías G, Segarra AB, Martínez-Cañamero M, Ramírez-Sánchez M, Prieto I. Influence of a virgin olive oil versus butter plus cholesterol-enriched diet on testicular enzymatic activities in adult male rats. Int J Mol Sci. 2017;18(8):1701. DOI: 10.3390/ijms18081701

Karp DR, Shimooku K, Lipsk PE. Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxida-tion-induced cell death. J Biol Chem. 2001;276:3798-804. DOI: 10.1074/jbc.M008484200

Tian WN, Braunstein LD, Apse K, Pang J, Rose M, Tian X, et al. Importance of glucose-6-phosphate dehydro-genase activity in cell death. Am J Physiol. 1999;276:1121-31. DOI: 10.1152/ajpcell.1999.276.5.C1121

Leopold JA, Zhang YY, Scribner AW, Stanton RC, Loscalzo J. Glucose-6-phosphate dehydrogenase overexpression de-creases endothelial cell oxidant stress and increases bioavailable nitric oxide. Arterioscler Thromb Vas Biol. 2003;23:411-7. DOI: 10.1161/01.ATV.0000056744.26901.BA

Poels J, Langendonckt A, Many MC, Wese FX, Wyns C. Vitrification preserves proliferation capacity in human spermatogonia. Hum Reprod. 2013;28(3):578-89. DOI: 10.1093/humrep/des455




How to Cite

Volkova N, Yuhta M, Sokil L, Chernyshenko L, Stepanuk L, Goltsev A. Efficiency of Combined Use of Fullerene C60 and Bovine Serum Albumin for Rehabilitation of Vitrified Fragments of Rat Immature Seminiferous Tubules. Innov Biosyst Bioeng [Internet]. 2021Sep.29 [cited 2021Dec.8];5(3):189-96. Available from: http://ibb.kpi.ua/article/view/241126