The effectiveness of biopolymers application for cryopreservation of the fragments of convoluted seminiferous tubules of prepubertal rat's testis

Home/2019, Vol. 7, No. 1/The effectiveness of biopolymers application for cryopreservation of the fragments of convoluted seminiferous tubules of prepubertal rat’s testis

Cell and Organ Transplantology. 2019; 7(1):12-17.
DOI: 10.22494/cot.v7i1.92

The effectiveness of biopolymers application for cryopreservation of the fragments of convoluted seminiferous tubules of prepubertal rat’s testis

Volkova N., Yukhta M., Chernyschenko L., Stepaniuk L., Sokil L., Goltsev A.
Institute of Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, Ukraine

Today, transplantation of cryopreserved fragments of immature testis is a non-alternative method of preserving the fertility of pre-adolescent patients who undergo cytotoxic therapy.
The purpose of the study is to compare the effectiveness of the use of biopolymers (bovine serum albumin and fibrin gel) as the bases of cryoprotective media at low temperature preservation of the fragments of the convoluted seminiferous tubules of prepubertal rats’ testis.
Materials and methods. Convoluted seminiferous tubules of prepubertal rats’ testis (75 ± 3 mg and 6-8 mm3), after a 30-minute exposure at 4 °C in cryoprotective media based on Hanks’ solution with 50 g/L of bovine serum albumin (BSA) or fibrin gel (FG) supplemented with 0.6 M DMSO or 0.7 M glycerol, were cryopreserved according to the program: 1 °C/min to -8 °C; stop for 10 minutes; 10 °C/min to -70 °C; stored in liquid nitrogen. After thawing, the histological structure was evaluated and the metabolic activity of the spermatogenic epithelium cells was determined.
Results. According to the results of the histological study, there was a positive tendency of FG application, which had the maximum expressiveness in combination with 0,7 M glycerol. In this case, 68.8 ± 15.7 % of cell nuclei remained morphologically intact, and changes in the spermatogenic epithelium were slightly pronounced. The metabolic activity of the rats’ seminiferous convoluted tubules cells after freezing and thawing remained at a significantly higher level when using FG in combination with 0.6 M DMSO than with 0.7 M glycerol compared to the corresponding cryoprotectant based on the Hanks’ solution with BSA.
Conclusions. The use of fibrin gel in the protocols of cryopreservation provide to preserve the histological structure and metabolic activity of the spermatogenic epithelium.

Key words: testicular tissue; prepubertal age; dimethyl sulfoxide; glycerol; cryopreservation; bovine serum albumin; fibrin gel

Full Text PDF (eng) Full Text PDF (ua)

1. Poels J, Abou-Ghannam G, Decamps A, et al. Transplantation of testicular tissue in alginate hydrogel loaded with VEGF nanoparticles improves spermatogonial recovery. J Control Release. 2016; 234:79-89. DOI:10.1016/j.jconrel.2016.05.037.
2. Kaneko H, Kikuchi K, Men NT, et al. Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice. Anim Sci J. 2018; 1-9. DOI: 10.1111/asj.13138.
3. Pukazhenthi BS, Nagashima J, Travis AJ, et al. Slow freezing, but not vitrification supports complete spermatogenesis in cryopreserved, neonatal sheep testicular xenografts. PLoS One. 2015; 10(4):e0123957. DOI: 10.1371/journal.pone.0123957.
PMid:25923660 PMCid:PMC4414479
4. Liu Z, Nie YH, Zhang CC, et al. Generation of macaques with sperm derived from juvenile monkey testicular xenografts. Cell Res. 2016; 26(1):139-142. DOI: 10.1038/cr.2015.112.
PMid:26369429 PMCid:PMC4816125
5. Shinohara T, Inoue K, Ogonuki N, et al. Birth of offspring following transplantation of cryopreserved immature testicular pieces and in vitro microinsemination. Hum Reprod. 2002; 17(12):3039-3045.
6. Ohta H, Wakayama T. Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally. Biol Reprod. 2005; 73(3):390-395. DOI: 10.1095/biolreprod.105.041673.
7. Lo KC, Yildiz C, Zhu Y, et al. Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero. J Urol. 2015; 194(2):585-91. DOI: 10.1016/j.juro.2015.01.099.
8. Poels J, Abou-Ghannam G, Herman S, et al. In search of better spermatogonial preservation by supplementation of cryopreserved human immature testicular tissue xenografts with N-acetylcysteine and testosterone. Front Surg. 2014; 1:47. DOI: 10.3389/fsurg.2014.00047.
PMid:25593971 PMCid:PMC4286969
9. Poels J, Van Langendonckt A, Many MC, et al. Vitrification preserves proliferation capacity in human spermatogonia. Hum Reprod. 2013; 28(3):578-589. DOI: 10.1093/humrep/des455.
10. Wyns C, Van Langendonckt A, Wese FX, et al. Long-term spermatogonial survival in cryopreserved and xenografted immature human testicular tissue. Hum Reprod. 2008; 23(11):2402-2414. DOI: 10.1093/humrep/den272.
11. Dafopoulos K, Griesinger G, Schultze-Mosgau A, et al. Cumulative pregnancy rate after ICSI with cryopreserved testicular tissue in non-obstructive azoospermia. Reprod Biomed Online. 2005; 10(4):461-6.
12. Picton HM, Wyns C, Anderson RA, et al. European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum Reprod. 2015; 30(11):2463-75. DOI: 10.1093/humrep/dev190.
13. Kanbar M, de Michele F, Wyns C. Cryostorage of testicular tissue and retransplantation of spermatogonial stem cells in the infertile male. Best Pract Res Clin Endocrinol Metab. 2018. pii: S1521-690X(18)30117-9. DOI: 10.1016/j.beem.2018.10.003.
14. Wyns C, Curaba M, Vanabelle B, et al. Options for fertility preservation in prepubertal boys. Hum Reprod Update. 2010; 16(3):312-328. DOI: 10.1093/humupd/dmp054.
15. Keros V, Hultenby K, Borgström B, et al. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Hum Reprod. 2007; 22(5):1384-1395. DOI: 10.1093/humrep/del508.
16. Milazzo JP, Travers A, Bironneau A, et al. Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining. J Androl. 2010; 31(6):617-630. DOI: 10.2164/jandrol.109.009324.
17. Jahnukainen K, Ehmcke J, Hergenrother SD, et al. Effect of cold storage and cryopreservation of immature non-human primate testicular tissue on spermatogonial stem cell potential in xenografts. Hum Reprod. 2007; 22(4):1060-1067. DOI: 10.1093/humrep/del471.
18. Onofre J, Baert Y, Faes K, et al. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. Hum Reprod Update. 2016; 22(6):744-761. DOI: 10.1093/humupd/dmw029.
PMid:27566839 PMCid:PMC5099994
19. Volkova NO, Yukhta MS, Chernyshenko LS, et al. Cryopreservation of rat seminiferous tubules using biopolymers and slow non-controlled rate cooling. Probl Cryobiol Cryomed 2018; 28(4):278-292. DOI: 10.15407/cryo28.04.278.
20. Miyamoto Y, Enosawa S, Takeuchi T, et al. Cryopreservation in situ of cell monolayers on collagen vitrigel membrane culture substrata: ready-to-use preparation of primary hepatocytes and ES cells. Cell Transplant. 2009; 18(5):619-626.
21. Volkova NO, Yukhta MS, Chernyshenko LG, et al. Exposure of seminiferous tubules of immature rats to cryoprotective media of various compositions. Probl Cryobiol Cryomed. 2017; 27(3): 203-218. DOI: 10.15407/cryo27.03.203.
22. Volkova N, Yukhta M, Goltsev A. Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats. Cell and tissue banking. 2018; 19(4):819-826. DOI: 10.1007/s10561-018-9740-z.
23. Campion SN, Carvallo FR, Chapin RE, et al. Comparative assessment of the timing of sexual maturation in male Wistar Han and Sprague-Dawley rats. Reprod Toxicol. 2013; 38(7):16-24.
24. Council of Europe [France]. European convention fort heprotection of vertebrate animals used for experimental and other scientific purposes. Strasbourg, 18.III.1986.
25. Milazzo JP, Vaudreuil L, Cauliez B, et al. Comparison of conditions for cryopreservation of testicular tissue from immature mice. Hum Reprod. 2008; 23(1):17-28. DOI: 10.1093/humrep/dem355.
26. Travers A, Milazzo JP, Perdrix A, et al. Assessment of freezing procedures for rat immature testicular tissue. Theriogenology. 2011; 76(6):981-990. DOI: 10.1016/j.theriogenology.2011.04.025.
27. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2):55-63.
28. Ogawa T, Arechaga JM, Avarbock MR, Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. International Journal of Developmental Biology. 2003; 41(1):111-122.
29. Grill G, Porcellini A, Lucarelli G. Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoetic stem cells. Cryobiology. 1980; 17(5):516-20.
30. Dimasi L. Meeting increased demands on cell-based processes by using defined media supplements. BioProcess International. 2011; 9(8):48-58.
31. Nishiyama K, Okudera T, Watanabe T, et al. Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects. Clin Exp Dent Res. 2016; 2(2):96-103. DOI: 10.1002/cre2.26.
PMid:29744155 PMCid:PMC5839250
32. Makarevich AV, Spalekova E, Olexikova L, et al. Effect of insulin-like growth factor I on functional parameters of ram cooled-stored spermatozoa. Zygote. 2014; 22(3):305-313. DOI: 10.1046/j.1365-2265.1998.00517.x.
33. Takahashi T, Hirsh A, Erbe E, et al. Mechanism of cryoprotection by extracellular polymeric solutes. Biophys J. 1988; 54(3):509-18. DOI: 10.1016/S0006-3495(88)82983-7.

Volkova N, Yukhta M, Chernyschenko L, Stepaniuk L, Sokil L, Goltsev A. The effectiveness of biopolymers application for cryopreservation of the fragments of convoluted seminiferous tubules of prepubertal rat’s testis. Cell and Organ Transplantology. 2019; 7(1):12-17. doi:10.22494/cot.v7i1.92

Creative Commons License
Is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.