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Vol. 24, Art. 46 (pp. 610-639)    |    2023       

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Experimental models for the study of male infertility in vivo and in vitro

Golubentseva Yu.V., Protasova G.A., Krasnyakova E.A., Kirilenko P.S., Popov V.B.

Federal State Unitary Enterprise "Scientific Research Institute of Hygiene, Occupational Pathology and Human Ecology" of the Federal Medical and Biological Agency

Brief summary

An experimental study of the problems of male reproduction, in particular, male infertility, and the search for approaches for the correction of pathological processes induced by toxicants in the spermatogenic epithelium of mouse testes were carried out. The aim of the work is to create two experimental models 1) male induced infertility in vivo - on the example of the action of the anthracycline antibiotic doxorubicin, which is used in the chemotherapy of a number of cancers and is a powerful cytotoxic agent, and 2) an in vitro model of development and maturation of spermatogenic cells from spermatogonia to mature spermatozoa. Both models can be used as objects of morphological and functional studies of the role of toxicants in the development of infertility, as sources of a possible pool of donor cells for compensation (correction) of toxic lesions, as an improvement in methods for testing chemical compounds for reprotoxicity. In experiments to create a model of male induced infertility in vivo, a single administration (i.v., i.p.) of doxorubicin to C57BL/6 or (C57BL/6×CBA) F1 mice was performed at doses of 6 or 10 mg/kg. Histological analysis of sections of the testes 9 days after exposure revealed the absence of spermatogonia in the tubules of the testes, an increase in the number of pycnotic cells, micronuclei, and vacuolization of the cytoplasm of Sertoli cells. By day 18, the absence of spermatogonia and spermatocytes was observed, hyperplasia of Sertoli cells was noted. By 27-36 days, the action of the drug led to the complete devastation of the seminiferous tubules, with the exception of Sertoli cells. Immunohistochemical staining of preparations on day 36 for the PLZF marker confirmed the presence of spermatogonial stem cells. Evaluation of the regenerative potential of the testes showed the appearance of spermatogonia and spermatocytes in part of the tubules on the 72nd day, most likely due to the activation of spermatogenic stem cells. Immunofluorescent analysis of the causes of cell death using the γH2AX biomarker revealed damage to the DNA of the testis suspension cells with an increase in the proportion of damaged nuclei on days 1-27. Analysis of the number and motility of epididymis spermatozoa in the period of 9-36 days after exposure revealed their sharp decrease by 36 days (by 3.9 times compared with control), a decrease in mobile forms to 11.8% versus 79.4% in control (p <0.001). In addition, the number of pathological forms of spermatozoa has slightly increased. The result of the work was the creation of two models for the study of induced male infertility. Their creation is aimed at developing methods for experimental correction (restoration) of testes with impaired reproduction of spermatogenic cells due to donor (autologous, allogeneic) transplantation of materials obtained in experiments in vivo and in vitro.


Key words

induction of male infertility; doxorubicin; correction; cultivation of spermatogenic cells

Reference list

1. Barratt C.L.R., Björndahl L., De Jonge C.J., et al. The diagnosis of male infertility: an analysis of the evidence to support the development of global WHO guidance?challenges and future research opportunities. Hum Reprod Update. 2017; 23:660-680.


2. Gamidov C., Avakyan A., Idiopaticheskoe besplodie y myjchin: epidemiologiya, etiologiya, patogenez, lechenie. Vrach. 2013; 7:2-4.


3. Kumar N., Singh A.K. Trends of Male Factor Infertility, an Important Cause of Infertility: A Review of Literature. J Hum Reprod Sci. 2015; 8:191-196. doi:10.4103/0974-1208.170370


4. Levine H., Jørgensen N., Martino-Andrade A., et al. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum Reprod Update. 2017; 23:646-659.


5. World Health Organization, WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. 2010, Cambridge University Press, Cambridge, UK, 5th edition


6. Kirilenko E.A. Empiricheskaya terapiya idiopaticheskogo besplodiya y myjchin. 2016: https://mcrz.rf/files/imb.pdf


7. Miyamoto T., Tsujimura A., Miyagawa Y., Koh E., et al. Male infertility and its causes in human. Advances in Urology, Male Infertility. Hindawi Publishing Corporation. 2011, 2012. doi: 10.1155/2012/384520.


8. Azizov A.P., Saidov M.S. Myjskoe besplodie. 2010; 51.


9. Triarico S., Rivetti S., Capozza M.A., et al. Transplacental Passage and Fetal Effects of Antineoplastic Treatment during Pregnancy. Cancers. 2022; 14(13): 3103.


10. Arnon J., Meirow D., Lewis-Roness H., Ornoy A. Genetic and teratogenic effects of cancer treatments on gametes and embryos. Human Reproduction Update. 2001; 7(4): 394-403.


11. Sharpe R.M. Environmental/lifestyle Effects on Spermatogenesis. Philos Trans R Soc London Ser B Biol Sci. 2010; 365(1546):1697-712.


12. Siu E.R, Mruk D.D, Porto C.S, Cheng C.Y. Cadmium-Induced Testicular Injury. Toxicol Appl Pharmacol. 2009; 238:240-249.


13. Baillie H.S., Pacey A.A., Moore H.D.M. Environmental chemicals and the threat to male fertility in mammals: evidence and perspective In Conservaion Biology 8. Reproductive Science and Integrated Conservation. Eds W.V Holt, A.R Pickard, JC Rodgers & DE Wilat. Cambridge, UK: Cambridge University Press ; 2003.


14. Komeya M., Kimura H., Nakamura H., et al. Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device. Sci Rep. 2016; 6: 21472.


15. McLean D.J. Spermatogonial stem cell transplantation and testicular function. Cell Tissue Res. 2005; 322: 21-31.


16. Wang X, Rivière I. genetic engineering and manufacturing of hematopoietic stem cells. Molecular Therapy: Methods & Clinical Development. 2017; 5: 94-105.


17. Richer G., Baert Y., Goossens E. In-vitro spermatogenesis through testis modelling: Toward the generation of testicular organoids Andrology. 2020; 8 (4): 879-891.


18. Stukenborg J.B., Schlatt S., Simoni M., et al. New horizons for in vitro spermatogenesis? An update on novel three-dimensional culturesystems as tools for meiotic and post-meiotic differentiation of testicular germ cells. Mol Hum Reprod. 2009; 15:521-529.


19. Choi N.Y., Park Y.S., Ryu J.S, Lee H.J. A novel feeder-free culture system for expansion of mouse spermatogonial stem cells. Mol. Cells 2014; 37(6): 473-479.


20. Pendergraft S.S., Sadri-Ardekani H., Atala A., Bishop C.E. Three-dimensional testicular organoid: a novel tool for the study of human spermatogenesis and gonadotoxicity in vitro. Biol Reprod. 2017; 96(3):720-732


21. Chapin R.E., Winton T., Nowland W. et al. Lost in translation: The search for an in vitro screen for spermatogenic toxicity. Birth Defects Res B. 2016; 107: 225-242.


22. Morgan S., Lopes F., Gourley C. et al. Cisplatin and doxorubicin induce distinct mechanisms of ovarian follicle loss; imatinib provides selective protection only against cisplatin. PLoS One. 2013; 8(7): 20117


23. Ben-Aharon I., Bar-Joseph H., Tzarfaty G. Doxorubicin-induced ovarian toxicity. Reproductive biology and endocrinology. 2010; 8:20.


24. Wang Q.L, Sun S.C, Han J. et al. Doxorubicin induces early embryo apoptosis by inhibiting poly(ADP ribose) polymerase. In Vivo. 2012; 26(5):827-34.


25. Akinjo O.O, Gant T.W, Marczylo E.L. Marczylo Perturbation of epigenetic processes by doxorubicin in the mouse testis. Toxicol. Res. 2016; 5: 1229-1243.


26. Wyrobek A.J., Bruce W.R. Chemical induction of sperm abnormalities in mice. Proc. Nat. Acad. Sci. USA. 1975; 72(11): 4425-4429.


27. Watanabe T., Endo A. Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice. Mutation Research. 1991; 262: 93-99.


28. Savchenkova I.P., Vasileva S.A. Kyltivirovanie spermatogonii hryaka na kletkah Sertoli. Citologiya. 2016; 58 (2): 135-142.


29. Volkova N.A., Korjikova S.V., Kotova T.O. i dr. Videlenie, kyltivirovanie i harakteristika spermatogoniev petyha. Selskohozyaistvennaya biologiya. 2013; 51 (4): 450-458.


30. Kaavya R., Kannan T.A., Basha S. H. Vairamuthu S., et al. Isolation of spermatogonial stem cell and its viability in mice by different enzymatic digestion methods. International J. of livestock research. 2016; 6(11): 54-60.


31. Oakberq E.F. Duration of spermatogenesis in the mouse and timing of stages of the cycle of the semiiniferous epithelium. Am J Anat. 1956; 99(3): 507-516.


32. Costoya J.A., Hobbs R.M., Barna M. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat. Genet. 2004; 36: 653-662.


33. Morrison AJ, Shen X. Chromatin remodelling beyond transcription: the INO80 and SWR1 complexes. Nat Rev Mol Cell Biol. 2009; 10(6): 373-84. doi: 10.1038/nrm2693.


34. Sakib S., Uchida A., Valenzuela-Leon P., Yu Y., et al. Formation of organotypic testicular organoids in microwell culture. Biology of Reproduction. 2019; 100(6): 1648-1660.


35. Nengzhuang W., Jiaming S., Minghua LIU, Long M.A, et al. A brief history of testicular organoids: from theory to the wards. 2022; 39(7):1423-1431.


36. Stukenborg J.-B., Jahnukainen K. In vitro spermatogenesis and its potential clinical implication for patients. in genetics of human infertility. Monogr Hum Genet., Vogt PH (ed), Basel, Karger. 2017; 21: 162-172.


37. Sato T., Katagiri K., Gohbara A., et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011; 471: 501-507.


38. Baert Y., Dvorakova-Hortova K., Margaryan H., Goossens E. Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds. Biofabrication. 2019; 11: 03501. doi: 10.1088/1758-5090/ab1452.


39. Tetsuhiro Y., Sato T., Katagiri K. In vitro spermatogenesis using an organ culture technique. In: Spermatogenesis: Methods and Protocols, Methods in Molecular Biology. 2013; 927: 479-88.


40. Baschat A.A., Schwinger E., Diedrich K. Debate. Assisted reproductive techniques. Are we avoiding the genetic issue? Hum. Reprod. 1996; 11:926-928.


41. Picton H.M., Wyns C., Anderson R.A., et al. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum Reprod. 2015; 30:2463-2475.