Background: Impairment of testicular function is one of complications of Diabetes mellitus (D.M) and this may result from increased oxidative stress. Vitamin E (Vit E) has antioxidant properties. Objective: To determine the microscopic changes in the diabetic testis with metformin alone or with Vit E and which of them are better to preserve the testicular structure. Materials and Methods: Fifty adult albino rats, were equally divided into five groups: Group 1: control group; group 2: diabetic group (diabetes was induced by (65 mg/kg; i.p) single dose of streptozotocin (STZ); group 3: vit.E-treated diabetic group (200 mg/kg/day) ip; group 4: Metformin-treated diabetic group (100 mg/kg/day) and group 5 Metformin and vit.E-treated diabetic group for eight weeks. Levels of testosterone, insulin and blood sugar were analyzed and testes specimens were prepared for light microscope and immunohistochemical detection of androgen receptor (AR) and caspase-3. Results: In diabetic group showed irregular seminiferous tubules with depletion, separation, vacuolation of the spermatogenic cells with perivascular and intertubular fibrosis also, the mean diameter of tubules significantly decreases. There are negative immunoexpressions of AR with positive caspase-3. Sections of metformin treated diabetic group showed incomplete or partial regeneration of germ cells while in group 4 and 5 showed almost complete regeneration of spermatogenic cells with increase in mean diameter of tubules, significantly increases in AR and decreases in caspase-3 expression. Conclusion: D.M produce damage in the testicular tissue and the concomitant administration of vit-E with metformin was shown better effect than metformin alone.
Diabetes mellitus is one of chronic common diseases and was taken as one of five causes of death [
Metformin is one of hypoglycemic drugs used in treatment of diabetes and its action done by increasing insulin sensitivity and allow easily passing of glucose in hyperglycemic state [
1) Drugs
Streptozotocin (diabetic drug) was purchased from Sigma-Aldrich, CHEMIE, GmbH, Germany; Lot No. SLBJ7785V. Vitamin E (±)-α-Tocopherol was purchased from Sigma-Aldrich, CHEMIE, GmbH, Germany EC No. 2334660, Lot No. RA20270; Metformin HCl; A free gift from Medical Union Pharmaceuticals Co. (MUP); Abosultan, Ismailia, Egypt, Batch No. 2014000594 Free Sample.
Streptozotocin was prepared freshly on the day of experiment. Vitamin E was dissolved and diluted in corn oil and was used in experiment [
2) Animals
Healthy, fifty adult male albino Wistar rats weighing 250 ± 20 g were purchased from the Animal House, Faculty of Medicine, Um Al-Qura University. The animals were kept under standard normal conditions of temperature (22˚C ± 2˚C), light (12 h light/12 h dark) and humidity (45%), for one week as an acclimatization period to accommodate the experiment conditions. During this period, animals were fed with a standard rat’s pellet diet and had free access to tap water ad libitum. All animal procedures experiment was done according to the standards for the care and use of laboratory animals, approved by the university scientific research ethics committee for experimental animals at Faculty of Medicine, Um Al-Qura University. They randomly equally divided into 5 groups and each group was housed in separate cages.
3) Induction of Diabetes Mellitus
Animals of group 2, 3, 4, 5 were overnight fasted and diabetes was induced by a single intraperitoneal (i.p) injection of Streptozotocin (STZ) in a dose of 65 mg/kg body weight dissolved in 0.1 M citrate buffer (pH 4.5) as freshly prepared according to [
4) Experimental Design
Fifty rats were randomly equally divided into 5 groups each consists of 10 rats and treated as following.
Group I: Normal control non diabetic rats, injected intraperitoneal (i.p) with the same volume of 0.1 M citrate buffer solution.
Group II: Control Diabetic rats, received daily oral dose of distilled water.
Group III: diabetic rats treated with metformin at a dose (100 mg/kg body weight/day) [
Group IV: Diabetic rats treated with Vitamin E at a dose (200 mg/kg body weight/day) [
Group V: Diabetic rats treated with combined metformin and Vitamin E at a doses of (100 mg/kg body weight/day) and (200 mg/kg body weight/day) respectively.
Treatments were administered once daily by oral gavage started at the 3rd day after induction of diabetes for eight weeks [
Then, overnight fasting rats were anesthetized with diethyl ether and blood samples were collected for various biochemical estimations from rat tail vein in which serum was separated by centrifugation at 2400 rpm for 20 min at 4˚C [
a) Body weight
Body weight was recorded 1 day before STZ-treatment and periodically every week during the study period and at the end of the study on overnight fasted animals.
b) Serum Blood Glucose and Insulin Concentrations
Fasting blood glucose level analysis was done by using glucometer (One Touch Basic, Lifescan J & J, USA). According to the manufacturer protocol serum insulin, was determined with Enzyme-Linked Immunosorbent Assay (ELISA) using specific commercial rat ELISA diagnostic kits.
c) Serum Testosterone, FSH and LH Levels
Serum testosterone, Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels were measured ELISA using specific commercial rat ELISA diagnostic kits according to the manufacturer protocol.
d) Serum Lipid Profile
Total Cholesterol (TC), serum Triglycerides (TG), High Density Lipoprotein-cholesterol (HDL-c), and low density lipoprotein-cholesterol (LDL-c) levels were measured photometrically by standard enzymatic calorimetric method, using commercial kits according to manufacturer instructions.
e) Testicular Redox Evaluation (In vivo antioxidant activity in testes homogenates)
In order to antithesis the rats they were injected by pentobarbital injection (60 mg/kg) [
f) Histological examination
After eight weeks of experiment, the weight of all animals were measured the rats weighted, and then anesthetized by pentobarbital injection (60 mg/kg) [
i) Hematoxylin & Eosin (H & E) for histological examinations [
ii) Masson’s trichrome stain for collagen fibers detections [
iii) Immunohistochemically studies to detect androgen receptors (AR) in which the polyclonal (rabbit) anti-ARs (PA1-111A; Affinity Bioreagents Inc., Colorado, Golden, USA) were used as the primary antibodies. Brown color reaction was detected in the nuclei of Sertoli cells, and Leydig cells. Counterstained with hematoxylin was done to all sections [
The (caspase-3) to examine the apoptotic cellular changes Caspase-3 antibody, which is a rabbit polyclonal antibody (CPP32) Ab-4 (Thermo Fisher Scientific, Fermont, California, USA). A standard avidin-biotin peroxidase complex system was used to detection of caspase-3 using according to the kit used (Neomarkers) (34) Sections were counterstained with hematoxylin [
iv) Evaluation of spermatogenetic activity: Johnson method was done for histological spermatogenetic score [
v) Morphometric study was done by using “OlympusBX40” image analyzer computer system (Cambridge, England) in Histology Department, Faculty of Medicine, Cairo University. Measurement and study of 10 non-over lapping fields for each animal at ×400 power to determine the following:
a. The mean seminiferous tubule diameter, were randomly examined and the tubule diameters of the ten most circular seminiferous tubules from each specimen were measured by using Sudaman method in which the average of the big and small diameter of tubule was calculated using the formula magnification [
b. The area % for the mission stained collagen fibers around seminiferous tubules.
c. The Area% for the immunohistochemical stains of caspase-3 and AR in the cells of the seminiferous tubules.
d. By a calibrated ocular lens of micrometer (Germany) on light microscope we calculation the number of Sertoli cells/tubule and number of STs/field and the number of Leydig cells/100 µm2.
g) Statistical analysis
All obtained data were expressed as (Mean ± SEM): One-way analysis of variance (ANOVA) was used to statistical analysis of collected data. p < 0.001 was considered statistically significant. Calculations were done on SPSS IBM, united states (version 16 windows) software [
The effects of different treatment on the rats’ body weight are illustrated in
Fasting blood glucose concentrations of the treated experimental animals at different time intervals during the experiment period are shown in
Final Body Weight (g) | Initial Body Weight (g) | GP |
---|---|---|
266.40 ± 3.11 | 255.00 ± 2.98 | Normal Control |
159.20 ± 3.09 | 257.40 ± 2.66 | Diabetic Control |
177.80 ± 2.69b | 254.60 ± 2.42 | Metformin |
249.60 ± 2.38a,b | 259.00 ± 3.27 | Vitamin E |
268.20 ± 1.86b | 252.60 ± 2.77 | Combination |
Values are expressed as means (±SEM). aNon-significant difference (p > 0.05) compared with control groups. bSignificant difference (p < 0.001) between treatment and control groups.
8 Weeks | 6 Weeks | 4 Weeks | 2 Weeks | 0 Week | Groups |
---|---|---|---|---|---|
94.60 ± 0.81 | 94.40 ± 0.93 | 93.20 ± 1.39 | 92.80 ± 0.73 | 93.80 ± 1.16 | Normal Control |
424.00 ± 2.00 | 434.60 ± 1.94 | 389.60 ± 5.11 | 345.00 ± 2.35 | 95.40 ± 0.75 | Diabetic Control |
122.80 ± 0.97b | 191.40 ± 1.97 | 234.40 ± 2.21 | 315.80 ± 1.36 | 97.20 ± 1.02 | Metformin |
169.60 ± 1.47b | 222.80 ± 2.56 | 279.20 ± 1.96 | 334.00 ± 1.92 | 95.80 ± 1.43 | Vitamin E |
101.20 ± 1.16b,a | 139.80 ± 1.50 | 214.20 ± 2.08 | 285.20 ± 2.48 | 94.60 ± 1.36 | Combination |
Values are expressed as means (±SEM). aNon-significant difference (p > 0.05) compared with control groups. bSignificant difference (p < 0.001) between treatment and control groups.
clear from the tabulated results that, in the normal control animals, treatment with the water alone did not significantly affect the blood glucose concentrations compared to the diabetic control group animals. While, the STZ-treated rats had increase in the blood glucose concentrations, and the values were significantly higher (p < 0.001). Rats treated with metformin showed a significant lowering of the blood glucose concentration (p < 0.001) compared to the diabetic rats. From the results, it is clear that treatment with vitamin E alone showed a marked decrease in blood glucose concentration (p < 0.001) compared to the diabetic rats. While, treatment with a combination of metformin and vitamin E showed the most effective decrease in the blood glucose concentration, compared to the diabetic rats and the treated groups, either metformin or the vitamin E alone. This result augments the hypothesis of the proposal, for the synergistic effects of metformin with vitamin E against STZ-induced diabetes, as the combination treatment, tended to nearly normalize the fasting blood glucose concentration, and there was no significant difference (p > 0.05) compared to the normal non-diabetic rats. STZ drug lead to reduce the serum insulin level compared to normal control rats. Treatment with metformin or vitamin E either alone or in combination, significantly (p < 0.001) increased the serum insulin levels compared to the non-treated diabetic rats
As shown in
8 Weeks | 0 Week | Groups |
---|---|---|
38.66 ± 0.37 | 38.64 ± 0.23 | Normal Control |
12.07 ± 0.26 | 38.93 ± 0.49 | Diabetic Control |
31.68 ± 0.44b | 38.66 ± 0.38 | Metformin |
29.69 ± 0.52b | 38.49 ± 0.27 | Vitamin E |
37.26 ± 0.33a,b | 38.94 ± 0.22 | Combination |
Values are expressed as means (±SEM). aNon-significant difference (p > 0.05) compared with control groups. bSignificant difference (p < 0.001) between treatment and control groups.
FSH (IU/L) | LH (IU/L) | Testosterone. (ng/ml) | GP |
---|---|---|---|
0.83 ± 0.01 | 0.58 | 3.59 ± 0.05 | Normal Control |
0.21 ± 0.03 | 0.18 | 0.53 ± 0.02 | Diabetic Control |
0.58 ± 0.03 | 0.38 | 1.83 ± 0.04 | Metformin |
0.55 ± 0.03 | 0.41 | 2.57 ± 0.05 | Vitamin E |
0.79 ± 0.04 | 0.53 | 3.41 ± 0.06 | Combination |
Values are expressed as means (±SEM) for all groups. aInsignificant difference (p > 0.05) compared with control groups. b,cSignificant difference (p < 0.001) in the same row between treatment and control groups.
Treatment with metformin or vitamin E alone partially increased the testosterone levels but not to the normal values, and there was significant difference (p < 0.001) compared to the normal control rats. While, treatment with a combination of vitamin E with metformin showed recovery of the FSH, LH and testosterone normal levels, and non-significant difference (p > 0.05) were obtained compared to the normal control rats, while a significant difference (p < 0.001) were obtained compared to the non-treated control diabetic rats.
From the results, it can be observed that, the untreated diabetic control rats were characterized by a significant difference in the biochemical parameters as total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c), and triglycerides (TG), compared to the normal control rats. While, diabetic rats showed a significant (p < 0.001) decrease in the high density lipoprotein-cholesterol (HDL-c) level in comparison to the normal control rats. Treatment with metformin normalize the biochemical parameters compared to the control diabetic rats. From the results, it could be observed that, vitamin E treatment almost tend to normalize the biochemical parameters in the treated rats and there is no-significant difference (p > 0.05) compared to the normal non-diabetic rats at the end of experiment, where rats in vitamin E and combination treated groups showed near normal levels of TC, TG, HDL-c, LDL-c.
The testicular antioxidant activity of CAT, SOD, and GSH in the STZ-treated
TC (mg/dL) | LDL-c (mg/dL) | HDL-c (mg/dL) | TG (mg/dL) | GP |
---|---|---|---|---|
93.37 | 65.58 | 49.15 | 61.81 | Normal Control |
194.03 | 144.14 | 21.37 | 155.48 | Diabetic Control |
143.52 | 98.63 | 32.06 | 97.23 | Metformin |
122.72 | 74.39 | 44.62 | 83.63 | Vitamin E |
104.37 | 68.15 | 47.82 | 72.24 | Combination |
Values are expressed as means (±SEM). aNon-significant difference (p > 0.05), compared with control groups. b,cSignificant difference (p < 0.001) between treatment and control groups.
CAT (mmple/min/mg protein) | MDA (nmol/mg protein) | GSH (mmole/g protein) | SOD (U/mg protein) | Groups/parameters |
---|---|---|---|---|
35.92b | 0.95a | 6.62 | 169.28 | Normal Control |
14.28 | 3.66 | 2.52 | 73.19 | Diabetic Control |
27.12 | 1.23 | 4.85 | 146.57 | Metformin |
30.24 | 1.02 | 5.83 | 151.16 | Vitamin E |
36.76 | 0.97 | 6.56 | 164.14 | Combination |
Values are expressed as means (±SEM). aNon-significant difference (p > 0.05), compared with control groups. b,cSignificant difference (p < 0.001) between treatment and control groups.
diabetic rats were significantly (p < 0.001) decreased, on the contrast, level of MDA was significantly (p < 0.001) increased compared to the normal control rats. The treatment of diabetic rats with metformin alone, significantly (p < 0.001) enhances the antioxidant parameters compared to the diabetic control rats. Treatment of diabetic rats with vitamin E alone led to a significant decrease in serum levels of lipid peroxidase (MDA), while, there are significant (p < 0.001) enhancement in the antioxidant enzyme activities (SOD, CAT and GSH) compared to the diabetic non-treated rats. While, treated diabetic rats with combination of metformin and vitamin E, showed normalization of the parameters, and there were significant (p < 0.001) difference compared to the diabetic non-treated rats.
H & E-stained sections of the testes of the control group showed many regular and rounded seminiferous tubules were separated by interstitial tissues which contain the polygonal Leydig cells with an acidophilic cytoplasm and tubules were lined by layers of spermatogenic cells and Sertoli cells. Sertoli cells were seen between spermatogenic cells resting on the basement membrane and have large ovoid nuclei (
In diabetic group, H&E-stained sections of the testes revealed irregularity of some seminiferous tubules and the spermatogenic cells were separated from their basement membrane with presence of sign of degeneration as vacuolation of their cytoplasm and there were few numbers of pale vacuolated interstitial cells of Leydig. There were wide interstitial spaces between the tubules with dilated blood vessels (
In vit-E treated diabetic group showed little number of seminiferous tubules had regular basement membrane and was lined by almost normal spermatogenic cells (
The mean area % of collagen fibers and mean area % of caspase-3 expression (
The mean diameter of seminiferous tubules in the diabetic group decreased in comparison to control and metformin treated diabetic rats (p < 0.05). In vit-E treated diabetic rats, the diameter of seminiferous tubules was decreased but this reduction was not significant in comparison with the control group while co-treatment with metformin significantly elevated in comparison with the control group (
Groups/parameters | Control group | Diabetic group | Diabetic + vit-E group | Diabetic + Metformin group | Diabetic + Metformin + vit ? E group |
---|---|---|---|---|---|
Mean area % | 0.16% | 15.68% | 5.77% | 3.67% | 0.22% |
SD± | 0.0427 | 4.1858 | 0.6103 | 3.7466 | 2.7967 |
p value | 0.15 | 0.212 | 0.03 | 0.126 | |
Significance | S | NS | S | S |
SD = standard deviation S = Significant at p < 0.05 NS= Non Significant
Groups/parameters | Control group | Diabetic group | Diabetic + vit - E group | Diabetic + Metformin group | Diabetic + Metformin + vit ? E group |
---|---|---|---|---|---|
Mean area % | 0 | 26.62 | 12.67 | 10.14 | 9.79 |
SD± | 0 | 3.1312 | 0.6010 | 2.0197 | 1.1006 |
p value | 0.05 | 0.134 | 0.002 | 0.05 | |
Significance | S | NS | S | S |
SD = standard deviation S = Significant at p < 0.05 NS= Non Significant
Groups/parameters | Control group | Diabetic group | Diabetic + vit - E group | Diabetic + Metformin group | Diabetic + Metformin + vit ? E group |
---|---|---|---|---|---|
ST Diameter (μm) (mean ± SEM) | 45.134 ± 4.352 | 37.04 ± 36.09a* | 42.01 ± 26.31a,b* | 39.60 ± 9.59a,b* | 44.40 ± 4.69a,b* |
Number of Leydig cells/100 µm2 | 22.31 ± 3.05 | 14.22 ± 1.75a* | 19.12 ± 3.70a,b* | 17.95 ± 8.67a,b* | 20.14 ± 6.79a,b* |
Number of Sertoli cells/tubule | 33.68 ± 2.96 | 19.16 ± 5.10a* | 26.95 ± 9.87a,b* | 25.75 ± 6.87a,b* | 29.95 ± 2.87a,b* |
Number of STs/field × 100 | 29.88 ± 2.61 | 11.88 ± 2.26a* | 25.35 ± 1.30a,b* | 27.21 ± 4.10a,b* | 30.33 ± 6.30a,b* |
Johnson score | 9.76 ± 0.42 | 5.47 ± 0.12a* | 6.93 ± 0.75a,b* | 8.74 ± 0.66a,b* | 9.50 ± 0.22a,b* |
Results are given as mean ± SEM. n = 10 for each group, *significant difference between control group and diabetic group: p < 0.05, Absence of subscript indicates no significant difference. A Significant difference vs. control group a. b Significant difference vs. diabetic group II.
of Leydig cells and Sertoli cells were significantly decreased (p < 0.05) in diabetic group (
We aimed in this study to compare the possible effects of metformin, or vitamin E or in combination with the vitamin E and metformin, on the testes of diabetic rats. Diabetes is one of chronic metabolic disease which leads to severe complications such as nephropathy, retinopathy and reproductive dysfunction [
From the results, there is no-significant difference (p > 0.05) in compared to the normal non-diabetic rats after 8 weeks’ treatment, where rats in vitamin E and combination treated groups showed near normal levels of TC, TG, HDL-c, LDL-c. All above results are as results in several studies [
The testicular antioxidant activity of CAT, SOD, and GSH in the STZ-treated diabetic rats was significantly (p < 0.001) decreased, on the contrast, level of MDA was significantly increased compared to the non-diabetic rats (p < 0.001). Our results are as previous studies, which showed that there was oxidative affection in diabetic patients in the form of increase testicular lipid peroxidation and decrease in testicular antioxidant mechanisms as CAT, SOD, and GSH activities [
Diabetes mellitus (DM) is lead to release of reactive oxygen species (ROS) which leads to oxidative stress, that is due to presence of chronic hyperglycaemia [
Results obtained from the present study revealed that using a combination of metformin and vitamin E was more effective in treatment of diabetic complications, especially on the reproductive system. So, antioxidants as vitamin E can be used as supporting protective therapy for reproductive dysfunction in diabetic patients and as potential novel therapeutic agents by diet.
The authors would like to thank Institute of Scientific Research and Revival of Islamic Heritage at Umm Al-Qura University, Makkah, KSA (Project No: 43509022) for the financial support.
The author declares no conflicts of interest.
Alasmari, W.A., Faruk, E.M., Abourehab, M.A.S., Elshazly, A.M.E. and El Sawy, N.A. (2018) The Effect of Metformin versus Vitamin E on the Testis of Adult Diabetic Albino Rats: Histological, Biochemical and Immunohistochemistry Study. Advances in Reproductive Sciences, 6, 113-132. https://doi.org/10.4236/arsci.2018.64010