THE FEATURES OF APOPTOSIS ACTIVATION IN SEMINIFEROUS TUBULE CELLS OF RATS WITH AZOOSPERMIA AFTER EXPOSURE OF BETA-RADIATION AT DOSES OF 2 AND 8 GRAY

The development of infertility in male rats under the influence of high doses of ionized radiation is caused not only by direct damage to cells, but also by the block of differentiation and the induction of apoptosis of maturing spermatogonia. However, the literature data on the activity of biological mediators of the internal and external pathways of apoptosis after irradiation with beta particles, in particular, with different effective doses, are extremely limited. Objective: to study the molecular biological mechanisms of apoptosis in the seminiferous tubules of male rats after irradiation with electrons at a dose of 2 and 8 Gray. Male Wistar rats (220±20 g; 9-10 weeks; n=40) were divided into 3 groups: Ist - control (n=10); IInd — electron irradiation, dose 2 Gray (n=15); IIIrd - electron irradiation, dose 8 Gray (n=15). The expression of mRNA of caspases CASP8 and CASP9 was determined on sections of testes with a thickness of 8 μm using real-time polymerase chain reaction. Apoptotic DNA fragmentation was detected by the TUNEL method with the calculation of TUNEL-positive nuclei. On the 50th day after the start of the experiment, a significant number of apoptotic cells were found in the groups of rats subjected to ionizing radiation. The relative number of TUNEL-positive cells of spermatogenic epithelium in the 8 Gray group was 56±1,2% compared with the 2 Gray group - 37±0,9% (p<0,01). In the control group, 10±1,1% TUNEL-positive cells were recorded. In the 8 Gray group, the expression of CASP9 was higher than in the 2 Gray group (5,83±0,48 versus 4,1±0,41 (p<0,01). The expression of CASP8 was not statistically significant between the control and experimental groups. In our experimental study, we obtained data indicating a dose-dependent effect of beta radiation on the induction of apoptosis in the seminiferous tubules of rats. It was found that the predominant activation of apoptosis after exposure to beta particles is due to an internal pathway with excessive activation of caspase-9 and, in all likelihood, is associated with a violation of the integrity and function of mitochondria.

1. Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis in humans. Fertil Steril. 2013;100(5):1180-1186. DOI:10.1016/j.fertnstert.2013.08.010

2. Gunes S, Al-Sadaan M, Agarwal A. Spermatogenesis, DNA damage and DNA repair mechanisms in male infertility. Reprod Biomed Online. 2015;31(3):309-319. DOI: 10.1016/j.rbmo.2015.06.010

3. Liu G, Gong P, Zhao H, Wang Z, Gong S, Cai L. Effect of low-level radiation on the death of male germ cells. Radiat Res. 2006;165(4):379-389. DOI:10.1667/rr3528.1

4. Abuelhija M, Weng CC, Shetty G, Meistrich ML. Rat models of post-irradiation recovery of spermatogenesis: interstrain differences. Andrology. 2013.1(2):206–215.

5. Zhang Z, Shao S, Meistrich ML. The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells. J Cell Physiol. 2007;211(1):149-158. DOI:10.1002/jcp.20910

6. Fang F, Gong PS, Zhao HG, et al. Mitochondrial modulation of apoptosis induced by low-dose radiation in mouse testicular cells. Biomed Environ Sci. 2013;26(10):820-830. DOI:10.3967/bes2013.005

7. Schmittgen T., Livak K. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101–1108.

8. Kasahara E, Sato EF, Miyoshi M, et al. Role of oxidative stress in germ cell apoptosis induced by di(2-ethylhexyl)phthalate. Biochem J. 2002;365(Pt 3):849-856. DOI:10.1042/BJ20020254

9. Amanpour P, Khodarahmi P, Salehipour M. Protective effects of vitamin E on cadmium-induced apoptosis in rat testes. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(3):349-358. DOI:10.1007/s00210-019-01736-w

10. Marouani N, Tebourbi O, Hallègue D, et al. Mechanisms of chromium hexavalent-induced apoptosis in rat testes. Toxicol Ind Health. 2017;33(2):97-106. DOI:10.1177/0748233715600333

11. Taneli F, Vatansever S, Ulman C, et al. The effect of spermatic vessel ligation on testicular nitric oxide levels and germ cell-specific apoptosis in rat testis. Acta Histochem. 2005;106(6):459-466. DOI:10.1016/j.acthis.2004.11.001

12. Hikim AP, Vera Y, Vernet D, et al. Involvement of nitric oxide-mediated intrinsic pathway signaling in age-related increase in germ cell apoptosis in male Brown-Norway rats. J Gerontol A Biol Sci Med Sci. 2005;60(6):702-708. DOI:10.1093/gerona/60.6.702

13. Estaquier J, Vallette F, Vayssiere JL, Mignotte B. The mitochondrial pathways of apoptosis. Adv Exp Med Biol. 2012;942:157-183. DOI:10.1007/978-94-007-2869-1_7

14. Silva AM, Correia S, Casalta-Lopes JE, et al. The protective effect of regucalcin against radiation-induced damage in testicular cells. Life Sci. 2016;164:31-41. DOI:10.1016/j.lfs.2016.09.003