Expression of testicular phosphorylated proteins in types 1 and 2 diabetes mellitus in mice: An experimental study


Background: Types 1 and 2 diabetes mellitus (DM) are known to be the cause of sub/infertility. However, the comparisons of potential markers in spermatogenesis and steroidogenesis in DM males have never been elucidated.

Objective: This study aimed to examine the expressions of tyrosine-phosphorylated and steroidogenic acute regulatory (StAR) proteins in testis of DM mice.

Materials and Methods: Fifty-six male C57BL/6 mice were divided into four groups (n = 14/ each): control of MLD-STZ (multiple low doses of streptozotocin), MLD-STZ, control of HFD-STZ (high-fat diet with STZ), and HFD-STZ. MLD-STZ mice (type 1 DM) were intraperitoneally (i.p.) injected with STZ at 40 mg/kg BW for five days. HFD-STZ mice (type 2 DM) received an HFD for 14 days and i.p.-induced by STZ at 85 mg/kg BW and fed with HFD. At the end of the experiment (days 36 and 72), the expressions of phosphorylated proteins and StAR were examined.

Results: Tyrosine phosphorylated proteins were localized in late spermatids, luminal fluid, and Leydig cells. The intensities of phosphorylated 110, 85, 72, 60, and 55 kDas were lower in the 36 day-DM mice. Although such intensities were present in both groups, only 85 kDa in the MLD-STZ mice was higher in HFD mice at 72 days. StAR expressions in both groups were decreased than that of the controls.

Conclusion: Decreased expressions of StAR and tyrosine-phosphorylated proteins may be directly involved in low testosterone levels and impaired spermatogenesis. These findings support the notion that both DM types play a role in male infertility.

[1] American Diabetes AssociationDiagnosis and classification of diabetes mellitus. Diabetes Care 2009; 32 (Suppl.): S62–67.

[2] Ahangarpour A, Oroojan AA, Heidari H, Ehsan G, Rashidi Nooshabadi MR. Effects of hydro-alcoholic extract of rhus coriaria (sumac) seeds on reproductive complications of nicotinamide-streptozotocin induced type-2 diabetes in male mice. World J Men’S Health 2014; 32: 151–158.

[3] Alves MG, Martins AD, Moreira PI, Carvalho RA, Sousa M, Barros A, et al. Metabolic fingerprints in testicular biopsies from type 1 diabetic patients. Cell Tissue Res 2015; 362: 431–440.

[4] Ballester J, Munoz MC, Dominguez J, Rigau T, Guinovart JJ, Rodriguez-Gil JE. Insulin-dependent diabetes affects testicular function by FSH- and LH-linked mechanisms. J Androl 2004; 25: 706–719.

[5] Sampannang A, Arun S, Sukhorum W, Burawat J, Nualkaew S, Maneenin C, et al. Antioxidant and hypoglycemic effects of Momordica cochinchinensis Spreng (Gac) aril extract on reproductive damages in streptozotocin (STZ)-induced hyperglycemia mice. Int J Morphol 2017; 35: 667–675.

[6] Sampannang A, Arun S, Burawat J, Sukhorum W, Iamsaard S. Testicular histopathology and phosphorylated protein changes in mice with diabetes induced by multiple-low doses of streptozotocin: An experimental study. Int J Reprod Biomed 2018; 16: 235–246.

[7] Xu Y, Lei H, Guan R, Gao Z, Li H, Wang L, et al. Studies on the mechanism of testicular dysfunction in the early stage of a streptozotocin induced diabetic rat model. Biochem Biophys Res Commun 2014; 450: 87–92.

[8] 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: 1–7.

[9] Zhang T, Zarkower D. DMRT proteins and coordination of mammalian spermatogenesis. Stem Cell Res 2017; 24: 195–202.

[10] Fu XF, Cheng SF,Wang LQ, Yin S, De Felici M, ShenW. DAZ family proteins, key players for germ cell development. Int J Biol Sci 2015; 11: 1226–1235.

[11] Kazarian E, Son H, Sapao P, Li W, Zhang Z, Strauss JF, et al. SPAG17 is required for male germ cell differentiation and fertility. Int J Mol Sci 2018; 19: 1252–1264.

[12] Arad-Dann H, Beller U, Haimovitch R, Gavrieli Y, Ben-Sasson SA. Immunohistochemistry of phosphotyrosine residues: identification of distinct intracellular patterns in epithelial and steroidogenic tissues. J Histochem Cytochem 1993; 41: 513–519.

[13] Chaichun A, Arun S, Burawat J, Kanla P, Iamsaard S. Localization and identification of tyrosine phosphorylated proteins in adult sprague-dawley rat testis. Int J Morphol 2017; 35: 1322–1327.

[14] Iamsaard S, Burawat J, Kanla P, Arun S, Sukhorum W, Sripanidkulchai B, et al. Antioxidant activity and protective effect of Clitoria ternatea flower extract on testicular damage induced by ketoconazole in rats. J Zhejiang Univ Sci B 2014; 15: 548–555.

[15] Arun S, Burawat J, Sukhorum W, Sampannang A, Maneenin C, Iamsaard S. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats. Int J Reprod Biomed 2016; 14: 443–452.

[16] Arun S, Burawat J, Sukhorum W, Sampannang A, Uabundit N, Iamsaard S. Changes of testicular phosphorylated proteins in response to restraint stress in male rats. J Zhejiang Univ Sci B 2016; 17: 21–29.

[17] Sukhorum W, Iamsaard S. Changes in testicular function proteins and sperm acrosome status in rats treated with valproic acid. Reprod Fertil Dev 2017; 29: 1585–1592.

[18] Shivaji S, Kumar V, Mitra K, Jha KN. Mammalian sperm capacitation: role of phosphotyrosine proteins. Soc Reprod Fertil Suppl 2007; 63: 295–312.

[19] Stival C, Puga Molina Ldel C, Paudel B, Buffone MG, Visconti PE, Krapf D. Sperm capacitation and acrosome reaction in mammalian sperm. Adv Anat Embryol Cell Biol 2016; 220: 93–106.

[20] Ventura-Sobrevilla J, Boone-Villa VD, Aguilar CN, Roma´ n-Ramos R, Vega-Avila E, Campos-Sepu´ lveda E, et al. Effect of varying dose and administration of streptozotocin on blood sugar in male CD1 mice. Proc West Pharmacol Soc 2011; 54: 5–9.

[21] Li X, Xu Z, Jiang Z, Sun L, Ji J, Miao J, et al. Hypoglycemic effect of catalpol on high-fat diet/streptozotocin-induced diabetic mice by increasing skeletal muscle mitochondrial biogenesis. Acta Biochim Biophys Sin 2014; 46: 738–748.

[22] Zhang M, Lv XY, Li J, Xu ZG, Chen L. The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model. Exp Diabetes Res 2008; 2008: 704045.

[23] Bose R, Adiga SK, D’Souza F, Salian SR, Uppangala S, Kalthur G, et al. Germ cell abnormalities in streptozotocin induced diabetic mice do not correlate with blood glucose level. J Assist Reprod Genet 2012; 29: 1405–1413.

[24] Gurley SB, Clare SE, Snow KP, Hu A, Meyer TW, Coffman TM. Impact of genetic background on nephropathy in diabetic mice. Am J Physiol Renal Physiol 2006; 290: 214–222.

[25] Chaudhry ZZ, Morris DL, Moss DR, Sims EK, Chiong Y, Kono T, et al. Streptozotocin is equally diabetogenic whether administered to fed or fasted mice. Lab Anim 2013; 47: 257–265.

[26] Liu CY, Hsu YJ, Chien YW, Cha TL, Tsao CW. Dietary resistant maltodextrin ameliorates testicular function and spermatogenesis in streptozotocin nicotinamide- induced diabetic rats. Andrologia 2016; 48: 363–373.

[27] Sa´ nchez-Garrido MA, Ruiz-Pino F, Manfredi-Lozano M, Leon S, Garcia-Galiano D, Casta˜no JP, et al. Obesity-induced hypogonadism in the male: premature reproductive neuroendocrine senescence and contribution of Kiss1-mediated mechanisms. Endocrinology 2014; 155: 1067–1079.

[28] Zhang Z, Yu Y, Xu H, Wang C, Ji M, Gu J, et al. High-fat diet aggravates 2,2’,4,4’-tetrabromodiphenyl ether-inhibited testosterone production viaDAX-1 in Leydig cells in rats. Toxicol Appl Pharmacol 2017; 323: 1–8.

[29] Liu GL, Zhang YM, Dai DZ, Ding MJ, Cong XD, Dai Y. Male hypogonadism induced by high fat diet and low dose streptozotocin is mediated by activated endoplasmic reticulum stress and IB and attenuated by argirein and valsartan. Eur J Pharmacol 2013; 713: 78–88.

[30] Richburg JH, Redenbach DM, Boekelheide K. Seminiferous tubule fluid secretion is a Sertoli cell microtubule-dependent process inhibited by 2,5-hexanedione exposure. Toxicol Appl Pharmacol 1994; 128: 302–309.