Effect of inorganic sodium nitrate on spermatozoid parameters, testicular miR-34b and miR-449a expression, apoptosis, and histological and stereological changes in type 1 diabetic male rat

Abstract

in this study the preventative and therapeutic effects of nitrate supplementation on the diabetes-induced testicular disorders in diabetic male rats were evaluated. Methods: Hundred male Wistar rats (6-7-weeks-old) weighting 85-115 g was used in this study. Animals were randomly divided into 10 groups (n=10/group) as follow: Control 2 months, Control 3 months, Control nitrate 2 months, Control nitrate 3 months, Diabetic 2 months, Diabetic 3 months, Diabetic insulin 2 months, Diabetic insulin 3 months, Diabetic nitrate 2 months, Diabetic nitrate 3 months. Sodium nitrate (100 mg/L nitrate in distilled drinking water) and NPH insulin (2 –4 U/day) was administered for two months; immediately after diabetes confirmation in 2 months groups and one month after diabetes confirmation in 3 months groups. Diabetes was induced by single 45 mg/kg intraperitoneal injection of streptozotocin. Blood glucose levels was measured on days 0, 3, 30, and 60 in 2 months groups and on days 0, 3, 30, 60, and 90 in 3 months groups. Body weight was recorded at the beginning and on days 0, 10, 40, and 60 in 2 month’s groups and on days 0, 10, 40, 60, and 90 in 3 months groups. Serum concentrations of insulin, LH, FSH, and testosterone were assessed using ELIZA kit. Serum level of NOx (NO metabolites) was determined by the Griess method. Real-time PCR was used for evaluating of miR-34b and miR-449a, and gene expressions of Pdcd4, Pacs2, and p53 in testis tissue. The testicular stereology, histopathology, apoptosis index and sperm parameters were assessed. Results: Induction of diabetes significantly decreased body weight, serum levels of insulin, NOx and testosterone, testis and sperm parameters, sperm motility, spermatogenesis index, Johnson’s score, miR-449a expression and testicular morphometric and stereological alterations. Whereas significantly increased blood glucose, cellular and tubular apoptosis index and testicular expression of miR-34b, P53, Pdcd4 and Pacs2. In both 2 and 3 months diabetic groups, nitrate supplementation significantly decreased blood glucose, expressions of miR-34b and P53, Pdcd4 and Pacs2, cellular and tubular apoptosis index as well as significantly increased body weight, serum insulin and NOx, Johnson’s score and miR-449a expression and improvement of testis and sperm parameters, morphometric and stereological alterations, spermatogenesis index and testis tissue structure were observed. The most of these effects were higher than those of insulin. A significant increase in serum testosterone level was seen just in 2 months diabetic rats receiving nitrate compared to the diabetic untreated group.