Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

Home/2021, Vol. 9, No. 1/Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

Cell and Organ Transplantology. 2021; 9(1):36-42.
DOI: 10.22494/cot.v9i1.120

Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells

Volkova N., Yukhta M., Sokil L., Chernyschenko L., Stepaniuk L., Goltsev A.

  • Institute for Problems of Cryobiology and Сryomedicine, National Academy of Sciences of Ukraine, Kharkiv, Ukraine

Abstract

The use of modern technologies of cryopreservation of testicular tissue samples in prepubertal patients is one of the ways to maintain their fertility in the future.
The purpose of the study was to investigate the proliferative potential, morphological characteristics and expression of specific markers of cell culture obtained from cryopreserved and vitrified fragments of seminiferous tubules (FSTs) of rats’ testis.
Materials and methods. The isolation of cells from native, cryopreserved and vitrified FSTs of immature rats was performed by incubation in a solution of collagenase type IV (1 mg/mL) + DNase (500 μg/mL). Cell viability was determined by Trypan blue staining. Monoclonal antibodies CD9-FITC, CD24-PE, CD45-FITC, CD90-FITC were used for immunophenotype analysis. Morphological characteristics, proliferative activity (MTT assay), relative number of cells positive for MAGE-B1 and vimentin were assessed in the obtained cultures.
Results. The analysis of phenotypic characteristics showed that cells from native, cryopreserved and vitrified FSTs were characterized by high expression level of CD9 (≥ 40 %), CD24 (≥ 70 %), CD90 (≥ 70 %) and low expression of the CD45 (≤ 1 %). In cell culture in vitro, the studied cells from cryopreserved and vitrified rat’s FSTs had the ability to adhere and proliferate while maintaining a cells population positive for MAGE-B1 and vimentin.
Conclusions. The results can be the basis for the development of effective protocols for the cultivation and cryopreservation of testicular spermatogonial stem cells in order to restore fertility in men.

Key words: testicular tissue; spermatogonial stem cells; cryopreservation

Full Text PDF

1. Song H, Wilkinson M. In vitro spermatogenesis a long journey to get tails. Spermatogenesis. 2012; 2(4):1-7. DOI: 10.4161/spmg.22069.
https://doi.org/10.4161/spmg.22069
PMid:23248764 PMCid:PMC3521745
2. Gohbara A, Katagiri K, Sato T, et al. In vitro murine spermatogenesis in an organ culture system. Biol Reprod. 2010; 83:261-267. DOI: 10.1095/biolreprod.110.083899.
https://doi.org/10.1095/biolreprod.110.083899
PMid:20393168
3. Uchida A, Dobrinski I. Germ cell transplantation and neospermatogenesis. Cham: Springer international publishing AG. 2018; 2018:361-375. DOI: 10.1007/978-3-319-42396-8_20.
https://doi.org/10.1007/978-3-319-42396-8_20
4. Goossens E, Tournaye H. Adult stem cells in the human testis. Semin Reprod Med. 2013; 31(1):39-48. DOI: 10.1055/s-0032-1331796.
https://doi.org/10.1055/s-0032-1331796
PMid:23329635
5. Smith JF, Yango P, Altman E, et al. Testicular niche required for human spermatogonial stem cell expansion. Stem Cells Transl Med. 2014; 3(9):1043-1054. DOI: 10.5966/sctm.2014-0045.
https://doi.org/10.5966/sctm.2014-0045
PMid:25038247 PMCid:PMC4149303
6. Yokonishi T, Sato T, Katagiri K, et al. In vitro spermatogenesis using anorgan culture technique. Methods Mol Biol. 2013; 927:479-488. DOI: 10.1007/978-1-62703-038-0_41.
https://doi.org/10.1007/978-1-62703-038-0_41
PMid:22992938
7. Mohaqiq M, Movahedin M, Mazaheri Z, et al. In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions. Biol Res. 2019; 52(1):16. DOI: 10.1186/s40659-019-0223-x.
https://doi.org/10.1186/s40659-019-0223-x
PMid:30917866 PMCid:PMC6438003
8. Sato T, Katagiri K, Kubota Y, et al. In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method. Nat Protoc. 2013; 8(11):2098-2104. DOI: 10.1038/nprot.2013.138.
https://doi.org/10.1038/nprot.2013.138
PMid:24091557
9. Baert Y, Braye A, Struijk RB, et al. Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell. Fertil Steril. 2015; 104(5):1244-1252. DOI: 10.1016/j.fertnstert.2015.07.1134.
https://doi.org/10.1016/j.fertnstert.2015.07.1134
PMid:26260199
10. Ginsburg ES, Yanushpolsky EH, Jackson KV. In vitro fertilization for cancer patients and survivors. Fertil Steril. 2001; 75(4):705-710. DOI: 10.1016/s0015-0282(00)01802-1.
https://doi.org/10.1016/S0015-0282(00)01802-1
11. 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):819-826. DOI:10.1007/s10561-018-9740-z
https://doi.org/10.1007/s10561-018-9740-z
PMid:30465307
12. Volkova NO, Yukhta MS, Chernyshenko LG, et al. Cryopreservation of rat seminiferous tubules using biopolymers and slow non-controlled rate cooling. Probl Cryobiol Cryomedicine. 2018; 28(4):278-292. DOI: 10.15407/cryo28.04.278.
https://doi.org/10.15407/cryo28.04.278
13. Volkova NO, Yukhta MS, Goltsev AM. Vitrification of rat testicular tissue using biopolymers. Biopolym Cell. 2020; 36(2):122-132. DOI: 10.7124/bc.000A26.
https://doi.org/10.7124/bc.000A26
14. Zhao Q, Caballero OL, Simpson AJG, Strausberg RL Differential evolution of MAGE genes based on expression pattern and selection pressure. PLoS ONE. 2012; 7(10):e48240. DOI.org/10.1371/journal.pone.0048240
https://doi.org/10.1371/journal.pone.0048240
PMid:23133577 PMCid:PMC3484994
15. Shinohara T, Avarbock MR, Brinster RL. β1-and α6-integrin are surface markers on mouse spermatogonial stem cells. PNAS. 1999; 96(10):5504-5509. DOI: 10.1073/pnas.96.10.5504.
https://doi.org/10.1073/pnas.96.10.5504
PMid:10318913 PMCid:PMC21889
16. Kubota H, Avarbock MR, Brinster RL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. PNAS. 2003; 100(11):6487-6492. DOI: 10.1073/pnas.0631767100.
https://doi.org/10.1073/pnas.0631767100
PMid:12738887 PMCid:PMC164473
17. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philosophical Transactions of the Royal Society B: Biol Sci. 2010; 365(1546):1663-1678. DOI: 10.1098/rstb.2010.0026.
https://doi.org/10.1098/rstb.2010.0026
PMid:20403877 PMCid:PMC2871929
18. Kanatsu-Shinohara M, Toyokuni S, Shinohara T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod. 2004; 70(1):70-75. DOI: 10.1095/biolreprod.103.020867.
https://doi.org/10.1095/biolreprod.103.020867
PMid:12954725
19. Ibtisham F, Honaramooz A. Spermatogonial Stem Cells for In Vitro Spermatogenesis and In Vivo Restoration of Fertility. Cells. 2020; 9(3):745. DOI: 10.3390/cells9030745.
https://doi.org/10.3390/cells9030745
PMid:32197440 PMCid:PMC7140722
20. Guan X, Chen P, Zhao X, et al. Characterization of stem cells associated with seminiferous tubule of adult rat testis for their potential to form Leydig cells. Stem cell Res. 2019; 41:101593. DOI: 10.1016/j.scr.2019.101593.
https://doi.org/10.1016/j.scr.2019.101593
PMid:31704538
21. Haack-Sorensen M, Bindslev L, Mortensen S, et al. The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use. Cytotherapy. 2007; 9(4):328-337. DOI: 10.1080/14653240701322235.
https://doi.org/10.1080/14653240701322235
PMid:17573608
22. Maleki M, Ghanbarvand F, Behvarz, MR, et al. Comparison of mesenchymal stem cell markers in multiple human adult stem cells. Int J Stem Cell. 2014; 7(2): 118. DOI: 10.15283/ijsc.2014.7.2.118.
https://doi.org/10.15283/ijsc.2014.7.2.118
PMid:25473449 PMCid:PMC4249894
23. Aponte PM, Soda T, Van De Kant HJG, de Rooij DG. Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor. Theriogenology. 2006; 65(9): 1828-1847. DOI:10.1016/j.theriogenology.2005.10.020
https://doi.org/10.1016/j.theriogenology.2005.10.020
PMid:16321433
24. Quintana M, Fon Tacer K, Hao YH, et al. Examining the temporal expression and regulation of type I MAGE proteins in spermatogenesis. FASEB J. 2014; 28(S1):930.
https://doi.org/10.1096/fasebj.28.1_supplement.930.4
25. Vogl AW, Vaid KS, Guttman JA. The Sertoli cell cytoskeleton. Adv Exp Med Biol. 2008; 636:186-211. DOI: 10.1007/978-0-387-09597-411.
https://doi.org/10.1007/978-0-387-09597-4_11
PMid:19856169
26. O’Donnell L, O’Bryan MK. Microtubules and spermatogenesis. Semin Cell Dev Biol. 2014; 30:45-54. DOI: 10.1016/j.semcdb.2014.01.003.
https://doi.org/10.1016/j.semcdb.2014.01.003
PMid:24440897
27. Wu S, Yan M, Ge R, et al. Crosstalk between sertoli and germ cells in male fertility. Trends Mol Med. 2020; 26(2):215-231. DOI: 10.1016/j.molmed.2019.09.006.
https://doi.org/10.1016/j.molmed.2019.09.006
PMid:31727542
28. Sharma S, Wistuba J, Pock T, et al. Spermatogonial stem cells: updates from specification to clinical relevance. Hum Reprod Update. 2019; 25(3):275-29. DOI: 10.1093/humupd/dmz006.
https://doi.org/10.1093/humupd/dmz006
PMid:30810745
29. Struijk RB, Mulder CL, Veen van der F, et al. Restoring fertility in sterile childhood cancer sur-vivors by autotransplanting spermatogonial stem cells: are we there yet? BioMed Res Int. 2013; 2013:903142. DOI: 10.1155/2013/903142.
https://doi.org/10.1155/2013/903142
PMid:23509797 PMCid:PMC3581117
30. Kanatsu-Shinohara M, Takashima S, Ishii K, et al. Dynamic changes in EPCAM expression during spermatogonial stem cell differentiation in the mouse testis. PloS One. 2011; 6(8):e23663. DOI: 10.1371/journal.pone.0023663.
https://doi.org/10.1371/journal.pone.0023663
PMid:21858196 PMCid:PMC3156235
31. Guo Y, Hai Y, Gong Y, et al. Characterization, isolation, and culture of mouse and human spermatogonial stem cells. J Cell Physiol. 2014; 229(4):407-413. DOI: 10.1002/jcp.24471.
https://doi.org/10.1002/jcp.24471
PMid:24114612
32. Hamra FK, Chapman KM, Nguyen DM, et al. Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. PNAS USA. 2005; 102:17430-17435. DOI: 10.1073/pnas.0508780102.
https://doi.org/10.1073/pnas.0508780102
PMid:16293688 PMCid:PMC1283987
33. Nagano MC. Techniques for culturing spermatogonial stem cells continue to improve. Biol Reprod. 2011; 84(1):5-6. DOI: 10.1095/biolreprod.110.088864.
https://doi.org/10.1095/biolreprod.110.088864
PMid:20944084
34. Guan K, Wagner S, Unsöld B, et al. Generation of functional cardiomyocytes from adult mouse spermatogonial stem cells. Circ Res. 2007; 100(11):1615-25. DOI: 10.1161/01.RES.0000269182.22798.d9.
https://doi.org/10.1161/01.RES.0000269182.22798.d9
PMid:17478732
35. Bojnordi MN, Azizi H, Skutella T, et al. Differentiation of Spermatogonia Stem Cells into Functional Mature Neurons Characterized with Differential Gene Expression. Mol Neurobiol. 2017; 54(7):5676-5682. DOI: 10.1007/s12035-016-0097-7.
https://doi.org/10.1007/s12035-016-0097-7
PMid:27644129
36. Hwang YS, Suzuki S, Seita Y. Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells. Nat commun. 2020; 11(1):1-17. DOI: 10.1038/s41467-020-19350-3.
https://doi.org/10.1038/s41467-020-19350-3
PMid:33168808 PMCid:PMC7653920

Volkova N, Yukhta M, Sokil L, Chernyschenko L, Stepaniuk L, Goltsev A.  Cryopreserved fragments of testicular seminiferous tubules of rats as a source of spermatogonial stem cells. Cell Organ Transpl. 2021; 9(1):36-42. doi:10.22494/cot.v9i1.120

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