Effect of sucrose on cryopreservation of pig spermatogonial stem cells

doi:10.1016/S2095-3119(16)61489-2

摘要/Abstract

Abstract: Sucrose is known to play an important role in the cryopreservation of sperm and female gonads; however, its effect on the cryopreservation of pig spermatogonial stem cells (pSSCs) has not been tested. The aim of this work was to study the effect of sucrose during pSSC cryopreservation and to find the most effective concentration in freezing medium. pSSCs were cryopreserved with freezing media containing different concentrations of sucrose (70, 140, 210, and 280 mmol L^–1) and a control group without sucrose. The survival rates, plasma membrane integrity, and mitochondrial membrane potential of thawed cells were detected by trypan blue (TB) staining, SYBR-14/propidium iodide (PI) dual staining, and JC-1 staining, respectively. All the staining results showed an obvious increase in cell survival in the sucrose-treated groups as compared to that in the control group, with the exception of 280 mmol L^–1 sucrose. Moreover, the 210 mmol L^–1 sucrose group yielded the highest survival rate among all the groups (P<0.05). The results of SYBR-14/PI dual staining and JC-1 staining were consistent with those of TB staining as above described. Quantitative real-time PCR (qRT-PCR) indicated that the mRNA levels of three apoptosis-promoting genes (BAX, APAF1 and CASPASE9) were significantly higher in thawed cells than in cells before freezing (P<0.05). Moreover, the mRNA level of one anti-apoptotic gene (XIAP) was significantly lower in thawed cells than in cells before freezing (P<0.05). When comparing the mRNA expression of apoptosis-related genes in thawed cells, the mRNA level of the anti-apoptotic genes in the control group was significantly lower than that in the sucrose-treated
groups (P<0.05). Western blot analyses showed that the expression levels of cleaved CASPASE9, CASPASE3 and PARP-1 in the sucrose-treated groups were lower than those in the control group and were the lowest in the 210 mmol L^–1 sucrose group. Both qRT-PCR and Western blot analyses suggested that sucrose inhibited cell apoptosis during freezing and thawing. Briefly, sucrose promoted pSSCs survival after freezing and thawing, especially at a concentration of 210 mmol L^–1, which possibly assisted pSSC dehydration and inhibited cell apoptosis. These findings hold great promise for further studies of the regulatory mechanism of proliferation and differentiation of pSSCs.

Key words: spermatogonial stem cells (SSCs), pig, cryopreservation, sucrose, apoptosis, slow-freezing

. [J]. Journal of Integrative Agriculture, 2017, 16(05): 1120-1129.

PAN Chuan-ying, YU Shuai, ZHANG Peng-fei, WANG Bo, ZHU Zhen-dong, LIU Ying-ying, ZENG Wen-xian . Effect of sucrose on cryopreservation of pig spermatogonial stem cells[J]. Journal of Integrative Agriculture, 2017, 16(05): 1120-1129.

参考文献

Aliakbari F, Yazdekhasti H, Abbasi M, Hajian Monfared M, Baazm M. 2016. Advances in cryopreservation of spermatogonial stem cells and restoration of male fertility. Microscopy Research and Technique, 79, 122–129.
Aponte P M, van Bragt M P, de Rooij D G, van Pelt A M. 2005. Spermatogonial stem cells: Characteristics and experimental possibilities. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 113, 727–742.
Avarbock M R, Brinster C J, Brinster R L. 1996. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Medicine, 2, 693–696.
Baert Y, Van Saen D, Haentjens P, In’t Veld P, Tournaye H, Goossens E. 2013. What is the best cryopreservation protocol for human testicular tissue banking? Human Reproduction, 28, 1816–1826.
Brinster R L, Avarbock M R. 1994. Germline transmission of donor haplotype following spermatogonial transplantation. Proceedings of the National Academy of Sciences of the United States of America, 91, 11303–11307.
Brinster R L, Zimmermann J W. 1994. Spermatogenesis following male germ-cell transplantation. Proceedings of the National Academy of Sciences of the United States of America, 91, 11298–11302.
Cai H, Wu J Y, An X L, Zhao X X, Wang Z Z, Tang B, Yue Z P, Li Z Y, Zhang X M. 2016. Enrichment and culture of spermatogonia from cryopreserved adult bovine testis tissue. Animal Reproduction Science, 166, 109–115.
Chaytor J L, Tokarew J M, Wu L K, Leclere M, Tam R Y, Capicciotti C J, Guolla L, von Moos E, Findlay C S, Allan D S, Ben R N. 2012. Inhibiting ice recrystallization and optimization of cell viability after cryopreservation. Glycobiology, 22, 123–133.
Critser J K, Huse-Benda A R, Aaker D V, Arneson B W, Ball G D. 1988. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertility and Sterility, 50, 314–320.
Duvigneau J C, Hartl R T, Groiss S, Gemeiner M. 2005. Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. Journal of Immunological Methods, 306, 16–27.
Garcia de Castro A, Tunnacliffe A. 2000. Intracellular trehalose improves osmotolerance but not desiccation tolerance in mammalian cells. FEBS Letters, 487, 199–202.
Goossens E, Tournaye H. 2014. Male fertility preservation, where are we in 2014? Annales d’Endocrinologie, 75, 115–117.
Gouk S S, Loh Y F, Kumar S D, Watson P F, Kuleshova L L. 2011. Cryopreservation of mouse testicular tissue: prospect for harvesting spermatogonial stem cells for fertility preservation. Fertility and Sterility, 95, 2399–2403.
Ha S J, Kim B G, Lee Y A, Kim Y H, Kim B J, Jung S E, Pang M G, Ryu B Y. 2016. Effect of antioxidants and apoptosis inhibitors on cryopreservation of murine germ cells enriched for spermatogonial stem cells. PLoS ONE, 11, e0161372.
Honaramooz A, Behboodi E, Megee S O, Overton S A, Galantino-Homer H, Echelard Y, Dobrinski I. 2003. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biology of Reproduction, 69, 1260–1264.
Iaffaldano N, Di Iorio M, Rosato M P. 2012. The cryoprotectant used, its concentration, and the equilibration time are critical for the successful cryopreservation of rabbit sperm: Dimethylacetamide versus dimethylsulfoxide. Theriogenology, 78, 1381–1389.
Izadyar F, Matthijs-Rijsenbilt J J, den Ouden K, Creemers L B, Woelders H, de Rooij D G. 2002. Development of a cryopreservation protocol for type A spermatogonia. Journal of Andrology, 23, 537–545.
Jiang Z, Rothschild M F. 2007. Swine genome science comes of age. International Journal of Biological Sciences, 3, 129–131.
Kanatsu-Shinohara M, Muneto T, Lee J, Takenaka M, Chuma S, Nakatsuji N, Horiuchi T, Shinohara T. 2008. Long-term culture of male germline stem cells from hamster testes. Biology of Reproduction, 78, 611–617.
Kanatsu-Shinohara M, Ogonuki N, Matoba S, Morimoto H, Ogura A, Shinohara T. 2014. Improved serum- and feeder-free culture of mouse germline stem cells. Biology of Reproduction, 91, 88.
Kanatsu-Shinohara M, Shinohara T. 2013. Spermatogonial stem cell self-renewal and development. Annual Review of Cell and Developmental Biology, 29, 163–187.
Kim K J, Lee Y A, Kim B J, Kim Y H, Kim B G, Kang H G, Jung S E, Choi S H, Schmidt J A, Ryu B Y. 2015. Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing. Cryobiology, 70, 175–183.
Lee K H, Lee W Y, Kim J H, Yoon M J, Kim N H, Kim J H, Uhm S J, Kim D H, Chung H J, Song H. 2013. Characterization of GFRα-1-positive and GFRα-1-negative spermatogonia in neonatal pig testis. Reproduction in Domestic Animals, 48, 954–960.
Lee W Y, Park H J, Lee R, Lee K H, Kim Y H, Ryu B Y, Kim N H, Kim J H, Kim J H, Moon S H, Park J K, Chung H J, Kim D H, Song H. 2013. Establishment and in vitro culture of porcine spermatogonial germ cells in low temperature culture conditions. Stem Cell Research, 11, 1234–1249.
Lee Y A, Kim Y H, Ha S J, Kim B J, Kim K J, Jung M S, Kim B G, Ryu B Y. 2014a. Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells. Fertility and Sterility, 101, 1165–1175.
Lee Y A, Kim Y H, Ha S J, Kim K J, Kim B J, Kim B G, Choi S H, Kim I C, Schmidt J A, Ryu B Y. 2014b. Cryopreservation of porcine spermatogonial stem cells by slow-freezing testis tissue in trehalose. Journal of Animal Science, 92, 984–995.
Lee Y A, Kim Y H, Kim B J, Jung M S, Auh J H, Seo J T, Park Y S, Lee S H, Ryu B Y. 2013. Cryopreservation of mouse spermatogonial stem cells in dimethylsulfoxide and polyethylene glycol. Biology of Reproduction, 89, 109.
Luo J, Megee S, Rathi R, Dobrinski I. 2006. Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: application to enrichment and culture of porcine spermatogonia. Molecular Reproduction and Development, 73, 1531–1540.
Manku G, Culty M. 2015. Mammalian gonocyte and spermatogonia differentiation: Recent advances and remaining challenges. Reproduction, 149, R139-R157.
Meryman H T. 1971. Cryoprotective agents. Cryobiology, 8, 173–183.
Mouttham L, Comizzoli P. 2016. The preservation of vital functions in cat ovarian tissues during vitrification depends more on the temperature of the cryoprotectant exposure than on the sucrose supplementation. Cryobiology, 73, 187–195.
Oldenhof H, Gojowsky M, Wang S, Henke S, Yu C, Rohn K, Wolkers W F, Sieme H. 2013. Osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. Biology of Reproduction, 88, 68.
Park M H, Park J E, Kim M S, Lee K Y, Park H J, Yun J I, Choi J H, Lee E, Lee S T. 2014. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. Journal of Assisted Reproduction and Genetics, 31, 983–991.
Patist A, Zoerb H. 2005. Preservation mechanisms of trehalose in food and biosystems. Colloids and Surfaces (B - Biointerfaces), 40, 107–113.
Pegg D E. 2007. Principles of cryopreservation. Methods in Molecular Biology, 368, 39–57.
Poels J, Abou-Ghannam G, Herman S, Van Langendonckt A, Wese F X, Wyns C. 2014. In search of better spermatogonial preservation by supplementation of cryopreserved human immature testicular tissue xenografts with N-acetylcysteine and testosterone. Frontiers in Surgery, 1, 47.
Sambu S. 2015. A Bayesian approach to optimizing cryopreservation protocols. PeerJ, 3, e1039.
Song H W, Wilkinson M F. 2014. Transcriptional control of spermatogonial maintenance and differentiation. Seminars in Cell & Developmental Biology, 30, 14–26.
Sum A K, Faller R, de Pablo J J. 2003. Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides. Biophysical Journal, 85, 2830–2844.
Tanpradit N, Comizzoli P, Srisuwatanasagul S, Chatdarong K. 2015. Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity. Theriogenology, 83, 1553–1561.
Tian T, Zhao G, Han D, Zhu K, Chen D, Zhang Z, Wei Z, Cao Y, Zhou P. 2015. Effects of vitrification cryopreservation on follicular morphology and stress relaxation behaviors of human ovarian tissues: Sucrose versus trehalose as the non-permeable protective agent. Human Reproduction, 30, 877–883.
Uchendu E E, Joachim Keller E R. 2016. Melatonin-loaded alginate beads improve cryopreservation of yam (Dioscorea alata and D. cayenensis). Cryo Letters, 37, 77–87.
Vuthiphandchai V, Nimrat S, Kotcharat S, Bart A N. 2007. Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores. Theriogenology, 68, 1192–1199.
Wang P, Li Y, Hu X C, Cai X L, Hou L P, Wang Y F, Hu J H, Li Q W, Suo L J, Fan Z G, Zhang B. 2014. Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells. Zygote, 22, 158–163.
Wu X, Goodyear S M, Abramowitz L K, Bartolomei M S, Tobias J W, Avarbock M R, Brinster R L. 2012. Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years. Human Reproduction, 27, 1249–1259.
Wu Y H, Shu J H, He C W, Li M, Wang Y J, Ou W B, He Y L. 2016. ROCK inhibitor Y27632 promotes proliferation and diminishes apoptosis of marmoset induced pluripotent stem cells by suppressing expression and activity of caspase 3. Theriogenology, 85, 302–314.
Wu Z, Falciatori I, Molyneux L A, Richardson T E, Chapman K M, Hamra F K. 2009. Spermatogonial culture medium: An effective and efficient nutrient mixture for culturing rat spermatogonial stem cells. Biology of Reproduction, 81, 77–86.
Yokonishi T, Ogawa T. 2016. Cryopreservation of testis tissues and in vitro spermatogenesis. Reproductive Medicine and Biology, 15, 21–28.
Zhang Y, Luo F, Wu S, Yu B, Liu T, Wu Y. 2014. Tribbles homolog 3 expression in spermatogonial stem cells of rat testes. Cell Biology International, 38, 1403–1407.
Zhao G, Takamatsu H, He X. 2014. The effect of solution nonideality on modeling transmembrane water transport and diffusion-limited intracellular ice formation during cryopreservation. Journal of Applied Physics, 115, doi: 10.1063/1.4870826
Zheng Y, He Y, An J, Qin J, Wang Y, Zhang Y, Tian X, Zeng W. 2014a. THY1 is a surface marker of porcine gonocytes. Reproduction Fertility and Development, 26, 533–539.
Zheng Y, Zhang Y, Qu R, He Y, Tian X, Zeng W. 2014b. Spermatogonial stem cells from domestic animals: Progress and prospects. Reproduction, 147, R65–R74.
Zohni K, Zhang X, Tan S L, Chan P, Nagano M C. 2012. The efficiency of male fertility restoration is dependent on the recovery kinetics of spermatogonial stem cells after cytotoxic treatment with busulfan in mice. Human Reproduction, 27, 44–53.