Animal Reproduction (AR)
Animal Reproduction (AR)

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation

Bruno Tomazele Rovani; Vitor Braga Rissi; Monique Tomazele Rovani; Bernardo Garziera Gasperin; Tadeu Baumhardt; Vilceu Bordignon; Liliane de Freitas Bauermann; Daniele Missio; Paulo Bayard Dias Gonçalves

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Radiotherapy causes destruction of tumor cells, but also threatens the integrity and survival of surrounding normal cells. Then, woman submitted to irradiation for cancer treatment may present permanent ovary damage, resulting in impaired fertility. The objective of this study was to investigate the effects of therapeutic doses of ionizing radiation (IR), used for ovarian cancer treatment in humans, on bovine cumulus-oocyte complexes (COCs) as experimental model. Bovine ovaries were exposed to 0.9 Gy, 1.8 Gy, 3.6 Gy or 18.6 Gy IR, and then COCs were collected and used to evaluate: (a) oocyte nuclear maturation; (b) presence of phosphorylated H2A.X (γH2AX), as an indicator of DNA double-strand breaks (DSBs); and (c) expression of genes involved in DNA repair (TP53BP1, RAD52, ATM, XRCC6 and XRCC5) and apoptosis (BAX). The radiation doses tested in this study had no detrimental effects on nuclear maturation and did not increase γH2AX in the oocytes. However, IR treatment altered the mRNA abundance of RAD52 (RAD52 homolog, DNA repair protein) and BAX (BCL2-associated X protein). We conclude that although IR doses had no apparent effect on oocyte nuclear maturation and DNA damage, molecular pathways involved in DNA repair and apoptosis were affected by IR exposure in cumulus cells.


ionizing radiation, infertility, oocyte, DNA damage


Adam-Guillermin C, Hertal-Aas T, Oughton D, Blanchard L, Alonzo F, Armant O, Horemans N. Radiosensitivity and transgenerational effects in non-human species. Ann ICRP. 2018;47(3-4):327-41. PMid:29745724.

Adriaens I, Smitz J, Jacquet P. The current knowledge on radiosensitivity of ovarian follicle development stages. Hum Reprod Update. 2009;15(3):359-77. PMid:19151106.

Ahmed MM, Ibrahim ZS, Alkafafy M, El-Shazly SA. L-Carnitine protects against testicular dysfunction caused by gamma irradiation in mice. Acta Histochem. 2014;116(6):1046-55. PMid:24925768.

Atun R, Jaffray DA, Barton MB, Bray F, Baumann M, Vikram B, Hanna TP, Knaul FM, Lievens Y, Lui TY, Milosevic M, O’Sullivan B, Rodin DL, Rosenblatt E, Van Dyk J, Yap ML, Zubizarreta E, Gospodarowicz M. Expanding global access to radiotherapy. Lancet Oncol. 2015;16(10):1153-86. PMid:26419354.

Baerwald AR. Human antral folliculogenesis: what we have learned from the bovine and equine models. Anim Reprod. 2009;6(1):20-9.

Barreta MH, Gasperin BG, Rissi VB, Cesaro MP, Ferreira R, Oliveira JF, Gonçalves PBD, Bordignon V. Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence. Exp Cell Res. 2012;318(16):2049-58. PMid:22691445.

Bettegowda A, Patel OV, Ireland JJ, Smith GW. Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, b-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, b-actin, and histone H2A during bovine oocyte maturation and early embryogenesis. Mol Reprod Dev. 2006;73(3):267–78. PMid: 16261607.

Beaugelin-Seiller K, Della-Vedova C, Garnier-Laplace J. Is non-human species radiosensitivity in the lab a good indicator of that in the field? Making the comparison more robust. J Environ Radioact. 2020;211:105870. PMid:30578084.

Chemaitilly W, Mertens AC, Mitby P, Whitton J, Stovall M, Yasui Y, Robison LL, Sklar CA. Acute ovarian failure in the childhood cancer survivor study. J Clin Endocrinol Metab. 2006;91(5):1723-8. PMid:16492690.

Chong MJ, Murray MR, Gosink EC, Russell HR, Srinivasan A, Kapsetaki M, Korsmeyer SJ, McKinnon PJ. Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system. Proc Natl Acad Sci USA. 2000;97(2):889-94. PMid:10639175.

De Felici M, Klinger FG. DNA Damage and apoptosis in fetal and ovarian reserve oocytes. In: Vázquez-Nin GH, Escobar ML, Felici M, Echeverría OM, Klinger FG, editors. Cell death in mammalian ovary. Dordrecht: Springer Springer; 2011. p. 143–63.

Ding M, Zhang E, He R, Wang X. Newly developed strategies for improving sensitivity to radiation by targeting signal pathways in cancer therapy. Cancer Sci. 2013;104(11):1401-10. PMid:23930697.

Firsanov DV, Solovjeva LV, Svetlova MP. H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues. Clin Epigenetics. 2011;2(2):283-97. PMid:22704343.

Gatei M, Sloper K, Sörensen C, Syljuäsen R, Falck J, Hobson K, Savage K, Lukas J, Zhou BB, Bartek J, Khanna KK. Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent Phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J Biol Chem. 2003;278(17):14806-11. PMid:12588868.

Ghardi M, Moreels M, Chatelain B, Chatelain C, Baatout S. Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells. Int J Mol Med. 2012;29(5):769-80. PMid:22322361.

Gong B, Chen Q, Endlich B, Mazumder S, Almasan A. Ionizing radiation-induced, Bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth Differ. 1999;10(7):491-502. PMid:10437917.

Gottardi FP, Mingoti GZ. Maturação de oócitos bovinos e influência na aquisição da competência para o desenvolvimento do embrião. Rev Bras Reprod Anim. 2010;33:82-94.

Hessels AC, Langendijk JA, Gawryszuk A, Heersters MAAM, van der Salm NLM, Tissing WJE, van der Weide HL, Maduro JH. Review – late toxicity of abdominal and pelvic radiotherapy for childhood cancer. Radiother Oncol. 2022;170:27-36. PMid:35257849.

Hiom K. DNA repair: Rad52 – the means to an end. Curr Biol. 1999;9(12):R446-8. PMid:10375523.

Hosseinimehr SJ. The protective effects of trace elements against side effects induced by ionizing radiation. Radiat Oncol J. 2015;33(2):66-74. PMid:26157675.

Jensen RB, Rothenberg E. Preserving genome integrity in human cells via DNA double-strand break repair. Mol Biol Cell. 2020;31(9):859-65. PMid:32286930.

Kocakavuk E, Anderson KJ, Varn FS, Johnson KC, Amin SB, Sulman EP, Lolkema MP, Barthel FP, Verhaak RGW. Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer. Nat Genet. 2021;53(7):1088-96. PMid:34045764.

Kumari S, Mukherjee S, Sinha D, Abdisalaam S, Krishnan S, Asaithamby A. Immunomodulatory effects of radiotherapy. Int J Mol Sci. 2020;21(21):8151. PMid:33142765.

Langbeen A, Porte HFM, Bartholomeus E, Leroy JLMR, Bols PEJ. Bovine in vitro reproduction models can contribute to the development of (female) fertility preservation strategies. Theriogenology. 2015;84(4):477-89. PMid:25981885.

Lei T, Du S, Peng Z, Chen L. Multifaceted regulation and functions of 53BP1 in NHEJ‑mediated DSB repair. Int J Mol Med. 2022;50(1):90. PMid:35583003.

Leibfried L, First NL. Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci. 1979;48(1):76-86. PMid:573253.

Levine JM, Whitton JA, Ginsberg JP, Green DM, Leisenring WM, Stovall M, Robison LL, Armstrong GT, Sklar CA. Nonsurgical premature menopause and reproductive implications in survivors of childhood cancer: a report from the childhood cancer survivor study. Cancer. 2018;124(5):1044-52. PMid:29338081.

Lo Presti A, Ruvolo G, Gancitano RA, Cittadini E. Ovarian function following radiation and chemotherapy for cancer. Eur J Obstet Gynecol Reprod Biol. 2004;113(Suppl 1):S33-40. PMid:15041128.

Lonati L, Barbieri S, Guardamagna I, Ottolenghi A, Baiocco G. Radiation-induced cell cycle perturbations: a computational tool validated with flow-cytometry data. Sci Rep. 2021;11:925.

Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature. 2012;481(7381):287-94. PMid:22258607.

MacPhail SH, Banáth JP, Yu TY, Chu EHM, Lambur H, Olive PL. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int J Radiat Biol. 2003;79(5):351-9. PMid:12943243.

Malhi PS, Adams GP, Singh J. Bovine model for the study of reproductive aging in women: follicular, luteal, and endocrine characteristics. Biol Reprod. 2005;73(1):45-53. PMid:15744017.

Marci R, Mallozzi M, Di Benedetto L, Schimberni M, Mossa S, Soave I, Palomba S, Caserta D. Radiations and female fertility. Reprod Biol Endocrinol. 2018;16(1):112. PMid:30553277.

Martin JH, Aitken RJ, Bromfield EG, Nixon B. DNA damage and repair in the female germline: contributions to ART. Hum Reprod Update. 2019;25(2):180-201. PMid:30541031.

Mavragani IV, Nikitaki Z, Kalospyros SA, Georgakilas AG. Ionizing radiation and complex DNA damage: from prediction to detection challenges and biological significance. Cancers. 2019;11(11):1789. PMid:31739493.

Meirow D, Biederman H, Anderson RA, Wallace WHB. Toxicity of chemotherapy and radiation on female reproduction. Clin Obstet Gynecol. 2010;53(4):727-39. PMid:21048440.

Ménézo Y, Dale B, Cohen M. DNA damage and repair in human oocytes and embryos: a review. Zygote. 2010;18(4):357-65. PMid:20663262.

Moscariello M, Wieloch R, Kurosawa A, Li F, Adachi N, Mladenov E, Iliakis G. Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining. DNA Repair. 2015;31:29-40. PMid:25973742.

Muñoz M, Santaballa A, Seguí MA, Beato C, Cruz S, Espinosa J, Fonseca PJ, Perez J, Quintanar T, Blasco A. SEOM clinical guideline of fertility preservation and reproduction in cancer patients. Clin Transl Oncol. 2016;18(12):1229-36. PMid:27896641.

Nikitaki Z, Hellweg CE, Georgakilas AG, Ravanat J-L. Stress-induced DNA damage biomarkers: applications and limitations. Front Chem. 2015;3:35. PMid:26082923.

Panganiban RA, Snow A, Day R. Mechanisms of radiation toxicity in transformed and non-transformed cells. Int J Mol Sci. 2013;14(8):15931-58. PMid:23912235.

Park MS. Expression of human RAD52 confers resistance to ionizing radiation in mammalian cells. J Biol Chem. 1995;270(26):15467-70. PMid:7797537.

Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys. 2004;59(4):928-42. PMid:15234026.

Pesty A, Doussau M, Lahaye JB, Lefèvre B. Whole-body or isolated ovary (60) Co irradiation: effects on in vivo and in vitro folliculogenesis and oocyte maturation. Reprod Toxicol. 2010;29(1):93-8. PMid:19874886.

Puy V, Barroca V, Messiaen S, Ménard V, Torres C, Devanand C, Moison D, Lewandowski D, Guerquin MJ, Martini E, Frydman N, Livera G. Mouse model of radiation-induced premature ovarian insufficiency reveals compromised oocyte quality: implications for fertility preservation. Reprod Biomed Online. 2021;43(5):799-809. PMid:34602345.

Rappold I, Iwabuchi K, Date T, Chen J. Tumor suppressor P53 binding protein 1 (53BP1) is involved in DNA damage–signaling pathways. J Cell Biol. 2001;153(3):613-20. PMid:11331310.

Ristic D, Modesti M, Kanaar R, Wyman C. Rad52 and Ku bind to different DNA structures produced early in double-strand break repair. Nucleic Acids Res. 2003;31(18):5229-37. PMid:12954758.

Rogakou EP, Boon C, Redon C, Bonner WM. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol. 1999;146(5):905-16. PMid:10477747.

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273(10):5858-68. PMid:9488723.

Ruiz-Herrera A, Garcia F, Garcia-Caldés M. Radiobiology and reproduction: what can we learn from mammalian females? Genes. 2012;3(3):521-44. PMid:24704983.

Schultz LB, Chehab NH, Malikzay A, Halazonetis TD. P53 binding protein 1 (53bp1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol. 2000;151(7):1381-90. PMid:11134068.

Shiloh Y, Ziv Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol. 2013;14(4):197-210.

Shim G, Normil MD, Testard I, Hempel WM, Ricoul M, Sabatier L. Comparison of individual radiosensitivity to γ-rays and carbon ions. Front Oncol. 2016;6:137. PMid:27379201.

Sirard MA. The ovarian follicle of cows as a model for human. In: Constantinescu G, Schatten H, editors. Animal models and human reproduction. Hoboken: John Wiley & Sons Inc.; 2017. p. 127-44.

Soares M, Sousa AP, Fernandes R, Ferreira AF, Almeida-Santos T, Ramalho-Santos J. Aging-related mitochondrial alterations in bovine oocytes. Theriogenology. 2020;157:218-25. PMid:32814249.

Sonmezer M, Oktay K. Fertility preservation in female patients. Hum Reprod Update. 2004;10(3):251-66. PMid:15140872.

Sudhakaran S, Uppangala S, Salian SR, Honguntikar SD, Nair R, Kalthur G, Adiga SK. Oocytes recovered after ovarian tissue slow freezing have impaired H2AX phosphorylation and functional competence. Reprod Fertil Dev. 2015;27(8):1242-8. PMid:25023890.

Tonorezos ES, Cohn RJ, Glaser AW, Lewin J, Poon E, Wakefield CE, Oeffinger KC. Long-term care for people treated for cancer during childhood and adolescence. Lancet. 2022;399(10344):1561-72. PMid:35430023.

Trenner A, Sartori AA. Harnessing DNA double-strand break repair for cancer treatment. Front Oncol. 2019;9:1388. PMid:31921645.

Vandersickel V, Mancini M, Slabbert J, Marras E, Thierens H, Perletti G, Vral A. The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons. Radiat Oncol. 2010;5(1):30. PMid:20423515.

Wallace WHB, Shalet SM, Crowne EC, Morris-Jones PH, Gattamaneni HR. Ovarian failure following abdominal irradiation in childhood: natural history and prognosis. Clin Oncol. 1989;1(2):75-9. PMid:2486484.

Wallace WHB, Thomson AB, Kelsey TW. The radiosensitivity of the human oocyte. Hum Reprod. 2003;18(1):117-21. PMid:12525451.

Wallace WHB, Thomson AB, Saran F, Kelsey TW. Predicting age of ovarian failure after radiation to a field that includes the ovaries. Int J Radiat Oncol Biol Phys. 2005;62(3):738-44. PMid:15936554.

Ward E, DeSantis C, Robbins A, Kohler B, Jemal A. Childhood and adolescent cancer statistics, 2014. CA Cancer J Clin. 2014;64(2):83-103. PMid:24488779.

Ward IM, Minn K, Van Deursen J, Chen J. p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. Mol Cell Biol. 2003;23(7):2556-63. PMid:12640136.

Weber AM, Ryan AJ. ATM and ATR as therapeutic targets in cancer. Pharmacol Ther. 2015;149:124-38. PMid:25512053.

Winship AL, Stringer JM, Liew SH, Hutt KJ. The importance of DNA repair for maintaining oocyte quality in response to anti-cancer treatments, environmental toxins and maternal ageing. Hum Reprod Update. 2018;24(2):119-34. PMid:29377997.

Yurut-Caloglu V, Caloglu M, Eskiocak S, Tastekin E, Ozen A, Kurkcu N, Oz-Puyan F, Kocak Z, Uzal C. Comparison of the protective roles of L-carnitine and amifostine against radiation-induced acute ovarian damage by histopathological and biochemical methods. J Cancer Res Ther. 2015;11(2):447-53. PMid:26148616.

Zhou G. Mechanisms underlying FLASH radiotherapy, a novel way to enlarge the differential responses to ionizing radiation between normal and tumor tissues. Radiat Med Protect. 2020;1(1):35-40.

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