Exogenous Testosterone and Vitamin E Ameliorates Diabetes-induced Necrosis of the Pancreas, Lipid and Testicular Disorders in Male Wistar Rats

Aims: To investigate the effect of combined administration of testosterone and vitamin E on diabetes-induced testicular dysfunctions and dyslipidemia in male Wistar rats. Study Design and Methodology: Fifty male Wistar rats (150-200 g) were randomly divided into ten groups of 5 animals: normal control (NC), normal-vit.E treated (NVE), normal-testosterone treated (NT), normal-vit.E+testosterone treated (NVET), diabetic-untreated (DU), diabetic-vit.E treated (DVE), diabetic-testosterone treated (DT), diabetic-vit.E + testosterone treated (DVET), diabetic-insulin treated (DI), and diabetic-glibenclamide treated (DG). Diabetes was induced with a single intraperitoneal injection of 120 mg/kg alloxan monohydrate. Rats with sustained blood glucose of ≥ 250 mg/dl were selected as diabetic. Blood glucose was measured using glucose/oxidase principle. Animals were treated for 14 days and by day 15, blood was collected from the retro-orbital plexus for serum lipid analysis. Testes, epididymis and pancreas were excised and caudal epididymal fluid was analyzed for spermatogenic indices while testes and pancreas were processed for histological evaluation. Data were expressed as mean±SEM and statistical analysis performed using Student's unpaired t test, one-way analysis of variance (ANOVA). Level of significance is P = .05. Results: The DVET showed significant reduction in serum TC (103.27±12.76), TG (76.25±9.79), VLDLc (15.25±1.96) and LDLc (69.70±14.74) compared with DU (188.67±9.97), (166.75±5.16), (33.55±1.12) and (141.16±11.06) respectively while significant increase was observed in DVET sperm count (59.60±10.31), sperm motility (74.00±2.19) and HDLc (18.31±2.22) compared with DU (25.20±3.99), (66.00±2.19) and (9.70±1.49) respectively. Conclusion: Combined administration of vit. E and testosterone ameliorates diabetes–induced dyslipidaemia, beta cells necrosis, reduced sperm count and motility in male Wistar rats.


INTRODUCTION
Diabetes mellitus is a metabolic disorder resulting from defect in insulin secretion, insulin action or both [1,2]. The defects in insulin availability have been reported to affect fertility in the male [3] and also cause disruption of the lipid profile [4]. La Vigneral et al. [5] reported that the absence or reduced stimulatory effect of insulin on the Leydig cell results in reduced testosterone production, atonia of seminal vesicles, bladder and urethra and increased oxidative stress. Reduced serum testosterone level [6] and increased oxidative stress [7] in diabetes have been reported to be closely linked to male infertility [8]. This study therefore investigated the combined effect of testosterone supplement and free radical scavenger (vitamin E) on the deleterious effect of diabetes on male reproduction.

Animal Care
Fifty healthy and normoglycaemic male Wistar rats having fasting blood glucose level of 70-80mg/dl and of average weight 172.5±25.00 g were used for this study. The animals were purchased from the animal house of the University of Ibadan, Ibadan. They were kept at normal room temperature and photo-periodicity of 12L: 12D in the animal house of the Faculty of Basic Medical Sciences and fed on rat pellets obtained from Vital Feeds Mill, Orogun, Ibadan. Water was made available ad libitum.

Induction of Diabetes
Diabetes was induced after an overnight fast by a single intra-peritoneal injection of alloxan monohydrate (120 mg/kg) [9]. Drop of blood from the dorsal vein of rats were used to determine the blood glucose levels at 12 hours, 24 hours, 36 hours, and 48 hours after the alloxan administration and throughout the study using the method of Majekodunmi et al. [10]. Rats with constant blood glucose level of 250 mg/dl or higher were selected into the diabetic group [11].

Experimental Procedure
Normal saline, vitamin E and glibenclamide were administered orally (P.O.) Testosterone and Insulin were administered intramuscularly (IM) ‫٭‬Testosterone (vials) was administered intramuscularly for three consecutive days and then every three days [12]. ‫٭‬Vitamin E was administered orally every three days [13].
Animals were treated for 14 days.

Preparation of Serum
About 4.0 ml of blood was withdrawn from the retro-orbital plexus of each rat into plain bottles and allowed to clot to obtain serum which was later centrifuged at 3500 rpm for 20 minutes. Clear serum was aspirated with a micropipette into new plane bottles and stored in refrigerator at 4°C for lipid profile analysis.

Lipid Profile Analysis
Total cholesterol (TC) and Triglycerides (TG) were estimated using the method of Rifai et al. [15], High Density Lipoprotein Cholesterol (HDLc) was analyzed using the method of Friedewald et al. [16]. Very Low Density Lipoprotein Cholesterol (VLDLc) and Low Density Lipoprotein Cholesterol (LDLc) were calculated from the values of TG, TC and HDLc by Friedelwald's equation [16].

Sperm Analysis
Animals were sacrificed by cervical dislocation, opened up at the abdomino-pelvic region and the caudal epididymis was excised and immersed in 5 ml formal-saline. The testes and pancreas were also excised for histological studies. The volume of fluid displaced by the caudal epididymis was taken as its volume. The caudal epididymis was homogenized in 5ml formalsaline to a suspension, and the spermatozoa were examined under the light microscope at a magnification of ×100. The sperm count was determined by evaluating five different fields on the improved Neubauer counting chamber (1/10 mm, LABART, Germany) [17], using the formula given below;
Eosin-Nigrosin stain (Sigma Co., USA) was used to identify dead sperm cells while live sperm cells were unstained. An hundred cells per slide were counted to obtain the percentage live/dead ratio [17] in determining the sperm viability.
Smear produced for sperm viability was retrieved and the slide was observed under the microscope with x100 objective under immersion oil; making a count of 400 spermatozoa and the spermatozoa abnormalities were classified as described by Badkoobeh et al. [17].

Histological Preparation
The testes and pancreas of the animals were excised, fixed and stained with H and E and processed for histological evaluation [19].

Statistical Analysis
Data are expressed as means±SEM. Statistical analysis was performed using Student's unpaired t test, one-way analysis of variance (ANOVA). P = .05 was considered statistically significant.

Blood Glucose (mg/dl)
There was no significant difference in the blood glucose level of normal rats treated with vit. E (106.80±3.92), normal rats treated with testosterone (106.80±2. 10

Sperm viability (% live spermatozoa)
There was no significant difference (p = .

Diabetic Untreated (DU)
No vascular congestion seen, pancreatic islets are shrunken, there was no visible lesion with acinar portions " Fig. 8".

Diabetic Vit. E treated (DVE)
The blood vessels are markedly congested, islets appear small, shrunken and with irregular outlines " Fig. 8".

Diabetic Testosterone treated (DT)
The blood vessels are markedly congested especially within the islets; exocrine portions appear normal; mild cytoplasmic vacuolation of cells of the islets " Fig. 8".

Diabetic Vit. E + Testosterone treated (DVET)
There is marked vacuolar change of the cells of the pancreatic acinar " Fig. 8".

Normal Control (NC)
Normal control group showed marked congestion of the blood vessels, numerous seminiferous tubules with normal regular outlines as well as adequate numbers of spermatogenic series (spermatogonia, spermatocytes and spermatids) " Fig. 9".

Diabetic Untreated (DU)
Diabetic untreated group showed a few seminiferous tubules with irregular outlines and depleted spermatogenic epithelium, devoid of late-spermatids with presence of abundant residual bodies " Fig. 9".

Diabetic Vit. E treated (DVE)
Diabetic vit. E treated group showed numerous closely-packed seminiferous tubules with regular outlines and abundant spermatogenic epithelium, early and late spermatids are abundant with presence of little amounts of residual bodies " Fig. 9".

Diabetic Testosterone treated (DT)
There are numerous closely-packed seminiferous tubules with regular outlines and moderately depleted spermatogenic epithelium. There are abundant late spermatids, abundant residual bodies, markedly congested blood vessels and accumulation of eosinophilic fluid around the seminiferous tubules " Fig. 10".

Diabetic Vit. E + Testosterone treated (DVET)
There are numerous closely-packed seminiferous tubules with regular outlines and abundant spermatogenic epithelium. Also present are abundant early and late spermatids, little amounts of residual bodies and mildly congested blood vessels " Fig. 10".

DISCUSSION
The significant increase in the blood glucose due to alloxan injection in the diabetic untreated rats (Table 1) agrees with the report of Szkudelski [9] in diabetes induction. The hyperglycemia and dyslipidemia observed in the diabetic untreated (Table 1, Figs. 2, 3 & 5) [11] could be due to reduced glucose uptake and utilization in muscles [20] typically observed in diabetes and reduced lipoprotein lipase activity in the liver [21]. The significant reduction in sperm count, sperm motility and significant increase in sperm middle piece abnormalities in the diabetic untreated rats could be due to high oxidative stress resulting from diabetes [22]. Reduced concentration of scavenging enzymes [22,23] has been reported to contribute to oxidative stress and this could also be responsible for the abnormalities observed in the sperm middle piece of the diabetic untreated rats.
The irregular outline of the islet and congested blood vessels which became conspicuous in diabetic group treated with vitamin E could possibly be the restorative effect of vitamin E compared to diabetic untreated (Fig. 8). The lipid profile in the diabetic vitamin E treated also showed significant reduction in total cholesterol, triglycerides, low density lipoprotein cholesterol, and very low density lipoprotein cholesterol (Figs. 1, 2, 3 & 4) which also confirm possible protective effect of vitamin E. Mendez and Balderaz [24] reported the protective effect of vitamin E on the membrane-bound lipoprotein lipase against lipid peroxidation and this may be the likely mechanism through which irregular outlines of the islets in the diabetic untreated was restored in the diabetic treated with vitamin E (Fig. 8). The significant increase in sperm count observed in the diabetic vitamin E treated (Fig. 6) could be attributed to the antioxidant effect of vitamin E which could have neutralized the free radicals as reported by Trabers and Steven [25] in a similar study.
Bal et al. [26] reported reduced testosterone in diabetes. Klentze [27] also reported that testosterone enhances the sensitivity of the peripheral tissues to insulin. In the testosterone treated diabetic group, a glycaemic reduction of 78.08% was observed and this could be a result of improved sensitivity of peripheral tissues to insulin and consequent utilization of blood glucose [27]. In addition, the involvement of peroxisomal PPAR-α receptor in fat metabolism [27,28] [26] and La Vigneral et al. [5] in which they reported significant reduction in the serum testosterone level in diabetics, it is most likely that the administered testosterone increased the plasma testosterone level, thereby increasing the stimulatory effect of testosterone not only on the peripheral tissues and peroxisomal PPAR-α receptor but also on the Sertoli cells with resultant significant increase in their sperm count and sperm motility (Figs. 6 & 7) [29,30].
The significant improvement observed in the lipid profile (Figs. 1-3), sperm count and sperm motility of the vitamin E + testosterone treated diabetic rats (Figs. 6 & 7) could be attributed to the combined antioxidant effect of vitamin E and the stimulatory effect of increased plasma testosterone.
Furthermore, the significant reduction in the sperm motility of the normal rats treated with vitamin E (Fig. 7) could be attributed to the oxidative stress effect of high α-tocopherol intake in unstressed individual [31]. Tilbrook and Clark [32] reported oxidative stress decreases axonemial protein phosphorylation and sperm mobilization which subsequently affect membrane fluidity and sperm motility. The significant reduction in the sperm motility observed in the normal rats treated with testosterone (NT) (Fig. 7), could be attributed to negative feedback effect of the administered testosterone on the hypothalamic -pituitarygonadal axis resulting in decreased testosterone secretion [21].
However, the significant decrease in sperm motility observed in the normal group treated with vitamin E + testosterone (Fig. 7) could be due to oxidative stress resulting from high concentration of vitamin E in circulation and the negative feedback effect of exogenously administered testosterone. Vitamin E levels in normal animals plus the administered vitamin E constituted high level of vitamin E which most likely resulted in oxidative stress. Again, the exogenous testosterone added to the normal level of testosterone in the normal animals could have resulted to a high dose of this hormone which could have invariably stimulated negative feedback mechanism through the hypothalamic -pituitary-gonadal axis. This could result in reduced testosterone secretion and plasma concentration. Combination of these effects i.e. the oxidative stress and reduced testosterone level could have resulted in reduced sperm motility which was observed in the study.

CONCLUSION
This study showed that combined administration of vitamin E and testosterone ameliorates diabetes-induced dyslipidaemia, beta cells necrosis, and reduced sperm count and sperm motility in male Wistar rats.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All authors hereby declare that "principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee.