Effects of 3, 4-Dihydroxyflavone on Cryopreserved Testicular Tissue of Neonatal Mouse

Afshin Pirnia, Abolfazl Zendedel, Abolfazl Abbaszadeh, Khatereh Anbari, Arman Mousavi, Mohammadreza Gholami*


Background and Aim: Infertility is a side effect of cancer treatment because of chemotherapy or radiotherapy. Cryopreservation of testicular tissue or spermatogonial stem cells before cancer treatment and their transplantation may preserve the natural fertility. Cryopreservation is a damaging process due to free radicals and toxic effect of frozen solution. The purpose of this paper is to study the antioxidant and antiapoptotic effects of 3',4'-dihydroxyflavone on the structure of frozen-thawed testicular tissue of neonatal mouse.

Materials and Methods: Testes of 6-day-old NMRI mice (N = 20) were isolated. Testicles were randomly divided into four groups: two groups as control groups for quick and slow freezing-melting process, and two others as treatment groups that underwent quick and slow freezing-melting with addition of 3',4'-dihydroxyflavone (10µM) in frozen solution. For the assessment of structural changes and apoptosis in the frozen-thawed testes, the hematoxylin-eosin staining and tunnel pod kits were used respectively. In order to analyze the data, we used Kruskal-Wallis Test and Mann-Whitney Test. The statistical analysis was entirely done by SPSS software.

Results: Histopathological changes and apoptosis rate were significantly decreased after thawing in both treatment groups in comparison with control groups.

Conclusion: Adding 3',4'-dihydroxyflavone (10µM) to freezing-melting environment and the use of quick freezing-melting method can reduce the histopathological and apoptotic changes.


Keywords: Neonatal mouse, Testicle, 3,4-dihydroxyflavone, Freezing-melting, oxidative

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Howlader N, Noone A, Krapcho M, Garshell J, Miller D, Altekruse S. SEER cancer statistics review, 1975-2012 [Internet]. Bethesda, MD: National Cancer Institute; 2013.[cited 2015 Jan 28].

Craft I, Tsirigotis M, Bennett V, Taranissi M, Khalifa Y, Hogewind G, et al. Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia. Fertility and sterility. 1995;63(5):1038-42.

Pirnia A, Parivar K, Hemadi M, Yaghmaei P, Gholami M. Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. Andrologia. 2016.

Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation. Biology of reproduction. 2003;68(3):1064-71.

Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proceedings of the National Academy of Sciences. 1994;91(24):11298-302.

Honaramooz A, Behboodi E, Blash S, Megee SO, Dobrinski I. Germ cell transplantation in goats. Mol Rep Develop. 2003;64(4):422-8.

Kim Y, Turner D, Nelson J, Dobrinski I, McEntee M, Travis AJ. Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog. Rep J. 2008;136(6):823-31.

Mikkola M, Sironen A, Kopp C, Taponen J, Sukura A, Vilkki J, et al. Transplantation of Normal Boar Testicular Cells Resulted in Complete Focal Spermatogenesis in a Boar Affected by the Immotile Short‐tail Sperm Defect. Reprod Domestic Anim. 2006;41(2):124-8.

Nagano M, McCarrey JR, Brinster RL. Primate spermatogonial stem cells colonize mouse testes. Biol Rep. 2001;64(5):1409-16.

Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nature Med. 2000;6(1):29-34.

Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proceedings of the National Academy of Sciences. 2001;98(11):6186-91.

Arendt J, Skene DJ. Melatonin as a chronobiotic. Sleep Med Rex views. 2005;9(1):25-39.

Fuller B, Paynter S. Fundamentals of cryobiology in reproductive medicine. Reproductive biomedicine online. 2004;9(6):680-91.

Pegg DE. Principles of cryopreservation. Cryopreservation and freeze-drying protocols. 2007:39-57.

Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification. Rep BioMed Online. 2004;9(2):164-70.

Sutton RL. Critical cooling rates for aqueous cryoprotectants in the presence of sugars and polysaccharides. Cryobiology. 1992;29(5):585-98.

Andreescu S, Hepel M. Oxidative Stress: Diagnostics, Prevention, and Therapy: American Chemical Society; 2011.

Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked? Free Radical Biol Med. 2010;49(11):1603-16.

Surh Y-J. Oxidative stress, inflammation, and health: CRC press; 2005.

Villamena FA. Molecular basis of oxidative stress: chemistry, mechanisms, and disease pathogenesis: John Wiley & Sons; 2013.

Umamaheswari S, Sangeetha KS. Anti-Inflammatory Effect of Selected Dihydroxyflavones. J clin Diagn Res: JCDR. 2015;9(5):FF05.

Thirugnanasambantham P, Viswanathan S, Mythirayee C, Krishnamurty V, Ramachandran S, Kameswaran L. Analgesic activity of certain flavone derivatives: a structure-activity study. J Ethnopharm. 1990;28(2):207-14.

Nambi RA, Viswanathan S, Thirugnanasambantham P, Reddy M, Dewan M, Sijher J, et al. Anti Inflammatory Activity Of Flavone and its Hydroxy Derivatives-A Structure Activity Study. Indian J Pharm Sci. 1996;58:18.

Jayashree B, Alam A, Vijay Kumar D, Nayak Y. Antioxidant and antibacterial activity of new 3-methylflavones. Indian J Heterocyclic Chem. 2010;19(3):237-40.

Wu E, Cole T, Davidson T, Blosser J, Borrelli A, Kinsolving C, et al. Flavones. 1. Synthesis and antihypertensive activity of (3-phenylflavonoxy) propanolamines without. beta.-adrenoceptor antagonism. J Med Chem. 1987;30(5):788-92.

Quan G-H, Chae H-S, Song HH, Ahn K-S, Lee H-K, Kim Y-H, et al. Anti-allergic flavones from Arthraxon hispidus. Chem Pharm Bulletin. 2013;61(9):920-6.

Cushnie TT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimic agents. 2005;26(5):343-56.

Wang TC, Chen YL, Tzeng CC, Liou SS, Chang YL, Teng CM. Antiplatelet α‐Methylidene‐γ‐butyrolactones: Synthesis and evaluation of quinoline, flavone, and xanthone derivatives. Helvetica Chimica acta. 1996;79(6):1620-6.

Lee KS, Kim EY, Jeon K, Cho SG, Han YJ, Yang BC, et al. 3, 4-Dihydroxyflavone acts as an antioxidant and antiapoptotic agent to support bovine embryo development in vitro. J Rep Develop. 2011;57(1):127-34.

Jung-Taek K, Sol-Ji P, Su-Jin K, Joon-Ho M, SAADELDIN IM, Goo J, et al. Effect of 7, 8-dihydroxyflavone as an antioxidant on in vitro maturation of oocytes and development of parthenogenetic embryos in pigs. J Rep Develop. 2013;59(5):450-6.

Gholami M, Saki G, Hemadi M, Khodadadi A, Mohammadi-asl J. Melatonin improves spermatogonial stem cells transplantation efficiency in azoospermic mice. Iran J Basic Med Sci. 2014;17(2):93.

Milazzo J, Vaudreuil L, Cauliez B, Gruel E, Massé L, Mousset-Siméon N, et al. Comparison of conditions for cryopreservation of testicular tissue from immature mice. Human Rep. 2008;23(1):17-28.

Abrishami M. Cryopreservation and xenografting of testis tissue. 2009.

Abrishami M, Anzar M, Yang Y, Honaramooz A. Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice. Theriogenology. 2010;73(1):86-96.

Bagchi A, Woods EJ, Critser JK. Cryopreservation and vitrification: recent advances in fertility preservation technologies. Expert Rev Med Dev. 2014.

Bank H, Brockbank K. Basic principles of cryobiology. J Cardiac Sur. 1987;2(1 Suppl):137-43.

Pegg DE. Cryopreservation of vascular endothelial cells as isolated cells and as monolayers. Cryobiology. 2002;44(1):46-53.

Kushki D, Azarnia M, Gholami M. Antioxidant Effects of Selenium on Seminiferous Tubules of Immature Mice Testis. Zahedan J Res Med Sci. 2015;17(12).

Zhang R, Kang KA, Piao MJ, Ko DO, Wang ZH, Chang WY, et al. Preventive effect of 7, 8-dihydroxyflavone against oxidative stress induced genotoxicity. Biological and Pharmaceutical Bulletin. 2009;32(2):166-71.

Jiang F, Guo N, Dusting GJ. Modulation of nicotinamide adenine dinucleotide phosphate oxidase expression and function by 3′, 4′-dihydroxyflavonol in phagocytic and vascular cells. J Pharm Exp Therap. 2008;324(1):261-9.

Choi J-Y, Kang J-T, Park S-J, Kim S-J, Moon J-H, Saadeldin IM, et al. Effect of 7, 8-dihydroxyflavone as an antioxidant on in vitro maturation of oocytes and development of parthenogenetic embryos in pigs. Rep Dev J. 2013;59(5):450-6.

Goto Y, Noda Y, Mori T, Nakano M. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radical Biol Med. 1993;15(1):69-75.

DOI: https://doi.org/10.22087/hmj.v1i2.593


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