International Journal of

increased percentage of tubules with positive SPI (Table II). More light microscopic analyses revealed that the germinal epithelium thickness significantly decreased in non-treated diabetic group. Meanwhile the treated animals were exhibited improved germinal epithelium thickness. Comparing the interstitial edema between different test groups showed that, the honey and metformin-simultaneous administration inhibited the diabetes-induced edema (Table II).
Texture studies showed that in C and HC groups, the seminiferous tubules and the germinal epithelium are completely healthy in terms of appearance and all levels of spermatogenic cells (Figure 1-A, 1-F). However, light microscopic analyses showed that the germinal epithelium of the diabetic rats underwent to a severe degeneration which was manifested with remarkable reduction in germ cells height. Moreover observations demonstrated that the germinal cells dissociation increased in diabetic rats which it was accomplished with dislocations in several tubules. Increased interstitial tissue expansion with remarkable edema between tubules observed in diabetes-induced rats.
Animals in MD and HMD groups were manifested with remarkable reduction in seminiferous tubules degeneration, increased germinal epithelium height and significant improvement in cellular junction (Figure1-C, 1-H, 1-E, 1-J). In diabetic group treated by honey (HD), all seminiferous tubules were normal with increased germinal epithelium height and improvement in cellular junction and all levels of spermatogenic cells (Figure 1-D, 1-I). Diabetic group that was treated by metformin (MD) also showed increased germinal epithelium height and seminiferous tubules compared to diabetic control group.



Table I. Effects of natural honey, Metformin or their co-administration on serum levels of testosterone, LH, FSH and Insulin in different groups (N=6 rats per group).





Table II. Effects of natural honey, Metformin or co-administration on seminiferous tubules diameter (STD), spermiogenesis index (SPI) and thickness of the epithelium in different groups (N=6 rats per group).





Figure 1: Cross section from testis; (A) control group: note normal testicular tissue with normal seminiferous tubules in higher magnification (F). (B) Non-treated diabetic testis: the seminiferous tubules are degenerated and remarkable edema is manifested in interstitial tissue. Note negative TDI and SPI in higher magnification (G). (C) Metformin and honey co-treated group, showing normal spermatogenesis (H) with decreased edema. (D) Honey alone-treated group: The tubules are appeared approximately normal accomplished with significant decrease in edema. Note the normal spermatogenesis in higher magnification (I). (E) Metformin alone-treated group: The tubules are presented with higher thickness of germinal epithelium (J) while the mild edema remained in interstitial tissue. H&E staining technique, (A, B, C, D, E: 100× and F, G, H, I, J: 400×).


Discussion
 
The deterioration of pancreatic β-cell function is caused by low expression of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase in the pancreas and increased generation of reactive oxygen species (ROS). Antioxidants have been identified as protection to the pancreas against oxidative stress in diabetes mellitus. Previously it has been shown that honey could fairly decrease the oxidative stress-induced damages in testicular tissue following smoking (11). Considering this finding, it may be concluded that the phenolic compounds such as flavonoids as a powerful antioxidants are able to prevent and/or inhibit diabetes-induced derangements (6).
On the other hand, honey contains different minerals such as zinc, selenium, copper, calcium, potassium, chromium, manganese. Some of these minerals have play vital roles in the maintenance of normal glucose tolerance, insulin secretion from the pancreas and some else involved in glucose and insulin metabolism (5). Thus here in this study the honey was administrated in order to protect the testicular tissue against diabetes-exerted damages in testicular tissue by two mechanisms of honey’s antioxidant feature and its effect on blood level of glucose. Present study showed that the blood level of insulin decreased in diabetic rats, which was in good accordance with those findings with Erejowa et al, whereas the diabetic animals which were received honey (HD) manifested with improved levels of insulin. This improvement could be related to honey’s protective effect on β-cells of pancreas (10).
It has been illustrated that, creating of any experimental diabetes in rats is associated with disorder in reproductive system physiologic function in both genders (18). Indeed, the diabetes impacts on the testicular tissue due to insufficient production of insulin which in turn results in reducing the sertoli and leydig cells endocrine function (19). Moreover the diabetes-decreased serum level of FSH can severely affect the testicular endocrine function and spermatogenesis as well. In the light of this hypothesis, previous reports showed that decreased level of insulin in diabetic rats resulted into remarkable reduction in FSH level (19).
Our biochemical analyses exhibited that the serum levels of FSH and LH decreased in diabetic rats. Meanwhile the rats in HD and HMD groups showed improved levels of gonadotrophic hormones. Minding that the insulin is essential for sustain receptors on leydig cells and for leydig cells division, we can suggest that honey alone (HD) and in combination with MF (HMD) could protect spermatogenesis by up-regulating the insulin level in the blood (20). This hypothesis approved by our biochemical analyses, as the animals in treated groups showed significantly increased levels of insulin.
Studies showed that oxidative stress reduces enzymatic and non-enzymatic level in leydig cells and causes reduction of testosterone. Therefore, the diabetes-induced oxidative stress in testicular tissue inhibits androgenesis by leydig cells (21). In present research the serum level of testosterone reduced in diabetic group which was in accordance with the findings of Stefanovic et al (22). We found that the animals in treated groups showed significant increased in testosterone level. As any derangement in testosterone synthesis can impact the spermatogenesis negatively, the honey, albeit with some differences, could up-regulate the testosterone level and subsequently protected spermatogenesis cell lineage (23).
It is well noted that there is a negative correlation between increased ROS level and normal spermatogenesis (24). It has been shown that the diabetes results in remarkable reduction in spermatogenesis and decreases the seminiferous tubules diameter by tubular atrophy (25). Histological observations revealed that the animals in HD and HMD groups manifested with positive spermiogenesis index.
Considering the fact that the normal spermatogenesis largely depends on lowered oxidative stress and increased endocrine activity by leydig and sertoli cells, we can conclude that honey can decrease tubular atrophy partly by down-regulating oxidative stress and as well by improving testosterone biosynthesis. Thus, the animals in treated groups exhibited remarkably higher germinal epithelium height and as well the seminiferous tubules diameter in comparison to non-treated animals.
 
Conclusion
 
Our data suggest that, the honey could inhibit the diabetes-induced damages in testicular tissue. Moreover, honey and metformin co-administration showed better results versus other forms of application. Thus it could be suggested that simultaneous administration of honey with metformin could be considered as appropriate form of application, as the testes of honey-received groups were manifested with improved histological features. Moreover, honey and metformin could improve testicular endocrine activities partly by regulating gonadotropins levels.
 
Acknowledgments
 
This research was financially supported by Ministry of Sciences and carried out at Urmia University.
 
Conflict of interest
 
All authors agreed to have the manuscript at present form and there are no conflict between authors and no conflicts of interests.