Histological investigation of the protective effect of metformin on testis and sperm parameters in obese rats with type 2 diabetes mellitus

Öz

Background/Aim: Diabetes mellitus (DM) is thought to have adverse effects on the male reproductive system. Metformin (MTF) is a widely used drug in the treatment of DM. This study hypothesized that MTF can reduce the harmful effects of DM on the male reproductive system in addition to diabetes treatment. Methods: Twenty-one adult (15-18 g, 11-12 months) C57BL6 male mice were randomly assigned to three groups, as follows: The Control group (C), Diabetes group (D), and Diabetes + Metformin (D+MTF) treatment group. Groups D and D+MTF were fed a diet composed of 60% fat for four weeks. DM was created by administering a single dose of 30 mg/kg streptozotocin intraperitoneally (i.p.). MTF was given through gavage at a dose of 300 mg/kg/day. At the end of the experiment, sperm parameters were evaluated in the testicular tissue. Histomorphology features and immune expressions of VEGF, Caspase-3, and Ki-67 were evaluated in testicular tissue sections stained with Hematoxylin and Eosin (HE). Results: Sperm concentration, motility, and morphological characteristics of diabetic mice were significantly reduced compared to control, and MTF-treated mice. Seminiferous tubule diameters and Johnsen scores were significantly higher in the control and D+MTF groups when compared to the diabetic group. VEGF and Ki-67 immune expression were significantly higher in the control and D+MTF groups compared to the D group (P=0.001, and P=0.002, respectively). Because of D, Caspase-3 positive cell density was higher in the D group compared to the control and D+MTF groups (P=0.002). Conclusion: Diabetes mellitus had adverse effects on sperm parameters and the testicular tissue. High glucose levels with decreased VEGF immune expression damaged spermatogenic cells and decreased proliferation and differentiation due to impaired intratesticular vascularity. MTF treatment reduced the damage in spermatogenic cells and contributed to the differentiation and division of cells through the restoration of vascularization.

Anahtar Kelimeler

• Metformin
• Type 2 Diabetes
• Obesity

Kaynakça

1. 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2008 Jan;31(Supplement 1):S55-S60. doi: 10.2337/dc08-S055
2. 2. Mealey BL, Ocampo GL. Diabetes mellitus and periodontal disease. Periodontol 2000. 2007;44:127-53.
3. 3. Agbaje IM, Rogers DA, McVicar CM, McClure N, Atkinson AB, Mallidis CL, et al. Insulin dependant diabetes mellitus: implications for male reproductive function. Hum Reprod. 2007;22(7):1871-7.
4. 4. Cai L, Chen S, Evans T, Deng, DX, Mukherjee K, Chakrabarti S. Apoptotic germ-cell death and testicular damage in experimental diabetes: prevention by endothelin antagonism. Urol Res. 2000;28(5):342-7.
5. 5. Shrilatha B, Muralidhara. Early oxidative stress in testis and epididymal sperm in streptozotocin-induced diabetic mice: its progression and genotoxic consequences. Reprod Toxicol. 2007;23(4):578-87.
6. 6. Kehler DS, Scott KD, Andrew NS, Shanel ES, Naomi CH, Dustin EK, et al. Cardiovascular complications of type 2 diabetes in youth. Biochem Cell Biol. 2015;93(5): 496-510.
7. 7. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989 December 8;246(4935):1306-9.
8. 8. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. Faseb J. 1999;13(1): 9-22.

9. 9. Caires KC, De Avila J, McLean DJ. Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis. Reprod. 2009;138(4):667-77.
10. 10. Rashid K, Sil PC. Curcumin ameliorates testicular damage in diabetic rats by suppressing cellular stress-mediated mitochondria and endoplasmic reticulum-dependent apoptotic death. Biochim Biophys Acta. 2015;1852(1):70-82.
11. 11. Chatterjee S, Khunti K, Davies MJ. Type 2 diabetes. Lancet. 2017;389(10085):2239-2251.
12. 12. Esteghamati A, Eskandari D, Mirmiranpour H, Noshad S, Mousavizadeh M, Hedayati M, et al. Effects of metformin on markers of oxidative stress and antioxidant reserve in patients with newly diagnosed type 2 diabetes: a randomized clinical trial. Clin Nutr. 2013;32(2):179-85.
13. 13. Attia SM, Helal GK, Alhaider AA. Assessment of genomic instability in normal and diabetic rats treated with metformin. Chem Biol Interact. 2009;180(2):296-304.
14. 14. Rabbani SI, Devi K, Khanam S. Role of Pioglitazone with Metformin or Glimepiride on Oxidative Stress-induced Nuclear Damage and Reproductive Toxicity in Diabetic Rats. Malays J Med Sci. 2010;17(1):3-11.
15. 15. Albasher G, Mona A, Mohamed M AD, Saad A, Rafa A. Protective effects of Artemisia judaica extract compared to metformin against hepatorenal injury in high-fat diet/streptozotocine-induced diabetic rats. Environ Sci Pollut Res Int. 2020;27(32):40525-36.
16. 16. Rangel SR, Evelyn NGS, Edgar EIF, Karine LR, Adriana RS, Cassiano F GA, et al. High-fat diet-induced kidney alterations in rats with metabolic syndrome: endothelial dysfunction and decreased antioxidant defense. Diabetes Metab Syndr Obes. 2019;12:1773-81.
17. 17. Zhang S, Huali X, XiaofengY, Yi W, Dayun S. Metformin ameliorates diabetic nephropathy in a rat model of low-dose streptozotocin-induced diabetes. Exp Ther Med. 2017;14(1):383-90.
18. 18. Yokoi K, Uthus EO, Nielsen FH. Nickel deficiency diminishes sperm quantity and movement in rats. Biol Trace Elem Res. 2003;93(1-3):141-54.
19. 19. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der MJP, Van Zyl AJ, et al. Sperm morphologic features as a prognostic factor in in-vitro fertilization. Fertil Steril. 1986;46(6):1118-23.
20. 20. Johnsen SG. Testicular biopsy score count a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones. 1970;1(1):2-25.
21. 21. Jalili C, Salahshoor M, Naderi T. The effect of hydroalcoholic extract of P. crispum on sperm parameters, testis tissue and serum nitric oxide levels in mice. Advanced Biomedical Research. 2015;4(1):40.
22. 22. Cameron DF, Rountree J, Schultz RE, Repetta D, Murray FT. Sustained hyperglycemia results in testicular dysfunction and reduced fertility potential in BBWOR diabetic rats. Am J Physiol. 1990;259(6 Pt 1):E881-9.
23. 23. Kushwaha S, Jena GB. Telmisartan ameliorates germ cell toxicity in the STZ-induced diabetic rat: studies on possible molecular mechanisms. Mutat Res. 2013;755(1):11-2.
24. 24. Kanter M, Aktas C, Erboga M. Curcumin attenuates testicular damage, apoptotic germ cell death, and oxidative stress in streptozotocin-induced diabetic rats. Mol Nutr Food Res. 2013;57(9):1578-85.
25. 25. Morgante G, Tosti C, Orvieto R, Musacchio MC, Piomboni P, De Leo V. Metformin improves semen characteristics of oligo-terato-asthenozoospermic men with metabolic syndrome. Fertil Steril. 2011;95(6):2150-2.
26. 26. Salahshoor MR, Mojdeh H, Shiva R, Fatemeh M, Cyrus J. Effect of Thymoquinone on Reproductive Parameter in Morphine-treated Male Mice. Adv Biomed Res. 2018;7:18.
27. 27. Zohreh N, Shahla R, Mahmoudi R. The Effect of Aloe Vera Extract on the Sperm Quality in Male Diabetic Rats. Bull. Env. Pharmacol. Life Sci. 2014;3:223-8.
28. 28. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182(3):311-22.
29. 29. Tsounapi P, Saito M, Fotios D, Koukos S, Shimizu S, Satoh K. et al. Antioxidant treatment with edaravone or taurine ameliorates diabetes-induced testicular dysfunction in the rat. Mol Cell Biochem. 2012;369(1-2):195-204.
30. 30. Cai L, Li W, Guangwu W, Luping G, Youchun J, James YK. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes. 2002;51(6):1938-48.
31. 31. Nna VU, AbuBakar AB, Azlina A, Mahaneem M. Diabetes-induced testicular oxidative stress, inflammation, and caspase-dependent apoptosis: the protective role of metformin. Arch Physiol Biochem. 2020;126(5):377-88.
32. 32. Ebisch IM, Thomas CMG, Wetzels AMM, Willemsen NP, Sweep FCGJ, Steegers-Theunissen RPM. Review of the role of the plasminogen activator system and vascular endothelial growth factor in subfertility. Fertil Steril. 2008;90(6):2340-50.
33. 33. Reddy N, Kasukurthi KB, Mahla RS, Pawar RM, Goelet S. Expression of vascular endothelial growth factor (VEGF) transcript and protein in the testis of several vertebrates, including endangered species. Theriogenology. 2012;77(3):608-14.
34. 34. Yoshida A, Anand-Apte B, Zetter BR. Differential endothelial migration and proliferation to basic fibroblast growth factor and vascular endothelial growth factor. Growth Factors. 1996;13(1-2):57-64.
35. 35. Sisman AR, Kiray M, Camsari UM, Evren M, Ates M, Baykara B, et al. Potential Novel Biomarkers for Diabetic Testicular Damage in Streptozotocin-Induced Diabetic Rats: Nerve Growth Factor Beta and Vascular Endothelial Growth Factor. Dis Markers. 2014;2014:108106. doi: 10.1155/2014/108106.