The effect of melatonin on the Bcl-2 and Bad proteins expression in ovarian corpus luteum cells after exposure to experimental hyperthermia

Background. There  is growing interest in  determining the  role of  melatonin in the regulation of proliferation and apoptosis of ovarian cells at various diseases and destabilizing influences. It is believed that the choice between the implementation of  a  cell  death or  survival program determines the  ratio of  anti-apoptotic and pro-apoptotic proteins.

The aim. To identify the effect ofmelatonin onthe expression ofanti-apoptotic Bcl-2 and pro-apoptotic Bad and the Bcl-2/Bad ratio in the ovarian luteocytes of Wistar rats in the acute (day 3) and recovery (days 7 and 14) periods after a single exposure to experimental hyperthermia.

Methods. Warming up took no  more than 17  minutes. Melatonin was injected subcutaneously (0.1 mg in 0.2 ml of physiological solution) for 3 days after experimental hyperthermia. Comparison groups included rats with physiological solution injection (control) and  animals after experimental hyperthermia + physiological solution injection. The Bad and Bcl-2 expression was determined immunohistochemically on days 3, 7 and 14 after experimental hyperthermia + physiological solution or melatonin injection.

Results. On the day 3 after experimental hyperthermia, the effect of the hormone was not detected. A week after experimental hyperthermia + melatonin injection, the Bad expression area decreased more significantly than in rats after experimental hyperthermia + physiological solution injection, which led to an increase in Bcl-2/ Bad ratio. This indicated an increase in anti-apoptotic protection, blocking the development of the internal apoptosis pathway at this time. 2 weeks after experimental hyperthermia + physiological solution injection, the Bcl-2 area decreased more significantly than the Bad area. As a result, the Bcl-2/Bad ratio decreased almost 2-fold compared to the control group. This indicated the activation of the “mitochondrial branch” of luteocyte apoptosis. 2 weeks after experimental hyperthermia + melatonin injection, the Bad and Bcl-2 areas decreased synchronously, which restored Bcl-2/ Bad to control values.

Conclusion. The  melatonin injection after experimental hyperthermia shifts the ratio of Bcl-2/Bad expression areas towards an increase in anti-apoptotic Bcl2 already a  week after the  recovery period and  promotes earlier normalization of Bcl-2/Bad to physiological levels (as early as 2 weeks after experimental hyperthermia + melatonin injection).

1. Bochkareva AL, Michurina SV, Konenkov VI, Bochkarev IG. The effect of melatonin on the follicular apparatus and vessels of the ovaries of rats in hyperthermia. Morphology. 2015; 148(5): 71-76. (In Russ.).

2. Duan L, Dong S, Huang K, Cong Y, Luo S, Zhang JZH. Computational analysis of binding free energies, hotspots and the binding mechanism of Bcl-xL/Bcl-2 binding to Bad/Bax. Phys Chem Chem Phys. 2021; 23(3): 2025-2037. doi: 10.1039/d0cp04693k

3. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: Recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 2018; 25(3): 486-541. doi: 10.1038/s41418-017-0012-4

4. Reiter RJ, Sharma R, Pires de Campos Zuccari DA, de Almeida Chuffa LG, Manucha W, Rodriguez C. Melatonin synthesis in and uptake by mitochondria: Implications for diseased cells with dysfunctional mitochondria. Future Med Chem. 2021; 13(4): 335-339. doi: 10.4155/fmc-2020-0326

5. Michurina SV, Kolesnikov SI, Ishchenko IY, Bochkareva AL, Arkhipov SA. Effect of melatonin on expression of apoptosis regulator proteins Bcl-2 and Bad in ovarian follicular apparatus after high temperature exposure. Bull Exp Biol Med. 2021; 170(5): 598-603. doi: 10.1007/s10517-021-05114-6

6. Michurina SV, Ishchenko IY, Arkhipov SA, Letyagin AY, Korolev MA, Zavjalov EL. The expression of apoptosis-regulating proteins Bcl-2 and Bad in liver cells of C57Bl/6 mice under lightinduced functional pinealectomy and after correction with melatonin. Vavilovskii Zhurnal Genet Selektsii. 2021; 25(3): 310-317. doi: 10.18699/VJ21.034

7. Yang C, Liu Q, Chen Y, Wang X, Ran Z, Fang F, et al. Melatonin delays ovarian aging in mice by slowing down the exhaustion of ovarian reserve. Commun Biol. 2021; 4(1): 534. doi: 10.1038/s42003-021-02042-z

8. Russo M, Forte G, Montanino Oliva M, Laganà AS, Unfer V. Melatonin and myo-inositol: supporting reproduction from the oocyte to birth. Int J Mol Sci. 2021; 22(16): 8433. doi: 10.3390/ijms22168433

9. Kvetnoy I, Ivanov D, Mironova E, Evsyukova I, Nasyrov R, Kvetnaia T, et al. Melatonin as the cornerstone of neuroimmunoendocrinology. Int J Mol Sci. 2022; 23(3): 1835. doi: 10.3390/ijms23031835

10. Zhang W, Wang Z, Zhang L, Zhang Z, Chen J, Chen W, et al. Melatonin stimulates the secretion of progesterone along with the expression of cholesterol side-chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) in corpus luteum of pregnant sows. Theriogenology. 2018; 108: 297-305. doi: 10.1016/j.theriogenology.2017.12.026

11. Pedreros M, Ratto M, Guerra M. Expression of functional melatonin MT(1) receptors in equine luteal cells: In vitro effects of melatonin on progesterone secretion. Reprod Fertil Dev. 2011; 23(3): 417-423. doi: 10.1071/RD10137

12. Zhang ZL, Peng J, Yang ST, Chen JX, Wang CX, Tong DW. Expression of arylalkylamine n-acetyltransferase (AANAT) and acetylserotonin o-methyltransferase (ASMT) in the corpus luteum of pregnant sows and synthesis of melatonin in luteal cells. Cell Tissue Res. 2022; 388(1): 167-179. doi: 10.1007/s00441-021-03556-y

13. Fang L, Li Y, Wang S, Yu Y, Li Y, Guo Y, et al. Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression. Aging (Albany NY). 2019; 11(20): 9013-9024. doi: 10.18632/aging.102367

14. Wang J, Zhu T, Ma X, Wang Y, Liu J, Li G, et al. Melatonergic systems of AANAT, melatonin, and its receptor MT2 in the corpus luteum are essential for reproductive success in mammals. Biol Reprod. 2021; 104(2): 430-444. doi: 10.1093/biolre/ioaa190

15. Ajayi AF, Akhigbe RE. Staging of the estrous cycle and induction of estrus in experimental rodents: An update. Fertil Res Pract. 2020; 6: 5. doi: 10.1186/s40738-020-00074-3

16. Efremov AV, Pakhomova YuV, Pakhomov EA, Ibragimov RSh, Shorina GN. Method of experimental modeling of general hyperthermia in small laboratory animals: Patent No. 2165105 C1 of the Russian Federation. 2001; (10). (In Russ.).

17. Yang L, Zhao Z, Cui M, Zhang L, Li Q. Melatonin restores the developmental competence of heat stressed porcine oocytes, and alters the expression of genes related to oocyte maturation. Animals (Basel). 2021; 11(4): 1086. doi: 10.3390/ani11041086

18. Arend LS, Knox RV. Fertility responses of melatonintreated gilts before and during the follicular and early luteal phases when there are different temperatures and lighting conditions in the housing area. Anim Reprod Sci. 2021; 230: 106769. doi: 10.1016/j.anireprosci.2021.106769

19. Bouroutzika E, Kouretas D, Papadopoulos S, Veskoukis AS, Theodosiadou E, Makri S, et al. Effects of melatonin administration to pregnant ewes under heat-stress conditions, in redox status and reproductive outcome. Antioxidants (Basel). 2020; 9(3): 266. doi: 10.3390/antiox9030266

20. Chen Y, Shan X, Jiang H, Guo Z. Exogenous melatonin directly and indirectly influences sheep oocytes. Front Vet Sci. 2022; 9: 903195. doi: 10.3389/fvets.2022.903195

21. Danilova MV, Usoltseva EN. Significance of the pineal gland hormone melatonin in maintaining the health of women of reproductive age (a review). Obstetrics, Gynecology and Reproduction. 2019; 13(4): 337-344. (In Russ.). doi: 10.17749/2313-7347.2019.13.4.337-344

22. Xing CH, Wang Y, Liu JC, Pan ZN, Zhang HL, Sun SC, et al. Melatonin reverses mitochondria dysfunction and oxidative stressinduced apoptosis of Sudan I-exposed mouse oocytes. Ecotoxicol Environ Saf. 2021; 225: 112783. doi: 10.1016/j.ecoenv.2021.112783

23. Ma J, Wang J, Hu S, Li Y, Zhang Y, Yang Y, et al. Effects of melatonin on development and hormone secretion of sheep theca cells in vitro. Theriogenology. 2023; 198: 172-182. doi: 10.1016/j.theriogenology.2022.12.036

24. Feng J, Ma WW, Li HX, Pei XY, Deng SL, Jia H, et al. Melatonin prevents cyclophosphamide-induced primordial follicle loss by inhibiting ovarian granulosa cell apoptosis and maintaining AMH expression. Front Endocrinol (Lausanne). 2022; 13: 895095. doi: 10.3389/fendo.2022.895095

25. Ma M, Chen XY, Li B, Li XT. Melatonin protects premature ovarian insufficiency induced by tripterygium glycosides: Role of SIRT1. Am J Transl Res. 2017; 9(4): 1580-1602.

26. Goktepe O, Balcioglu E, Baran M, Cengiz O, Ceyhan A, Suna PA, et al. Protective effects of melatonin on female rat ovary treated with nonylphenol. Biotech Histochem. 2023; 98(1): 13-19. doi: 10.1080/10520295.2022.2075566

27. Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L, et al. Melatonin as a free radical scavenger in the ovarian follicle. Endocr J. 2013; 60(1): 1-13. doi: 10.1507/endocrj.ej12-0263

28. He C, Ma T, Shi J, Zhang Z, Wang J, Zhu K, et al. Melatonin and its receptor MT1 are involved in the downstream reaction to luteinizing hormone and participate in the regulation of luteinization in different species. J Pineal Res. 2016; 61(3): 279-290. doi: 10.1111/jpi.12345

29. Wang X, Meng K, He Y, Wang H, Zhang Y, Quan F. Melatonin stimulates STAR expression and progesterone production via activation of the PI3K/AKT pathway in bovine theca cells. Int J Biol Sci. 2019; 15(2): 404-415. doi: 10.7150/ijbs.27912