DM is a metabolic disease that poses a serious risk to public health and causes severe damage to a number of organs. This disease also regards as one of the important cause of morbidity and mortality worldwide Moreover, growing evidence suggests that DM is closely associated with fertility problems, particularly in the male [29]. In fact, previous clinical and pre-clinical studies have reported that DM causes serious disruptions in male reproductive function by paving the way for problems such as testicular tissue defects [29, 30]. Similar to the literature, the results of our histopathological examination (JTBS) revealed that DM was dramatically disrupted histo-architecture of the testicular tissue and caused histopathological alterations such as seminiferous tubular atrophy, tubular degeneration, interstitial edema and Leydig cells losses. Another facts frequently reported in studies examining the effects of diabetes on male infertility is that DM causes a decrease in testosterone level and total epididymal sperm count [31–35]. Taken together with literature, it can be argued that Leydig cell losses are clearly associated with the decrease in testosterone and total epididymal sperm count in diabetic rats.
In addition to testicular tissue defects, DM can triggers severe sperm morphology abnormalities by increasing the oxidative stress in the male reproduction system. DM-induced oxidative stress causes LPO in mammalian sperm, which contains abundant unsaturated fatty acids in their membranes [36]. Also, oxidative stress causes an increase in LPO products such as MDA and a decrease in activation of antioxidant enzymes such as SOD [37–39]. More importantly, LPO triggers sperm plasma membrane degeneration and DNA fragmentation and causes sperm morphology abnormalities in mammals [9]. In this study, we found that DM caused SOD activity to decrease but MDA levels and abnormal sperm morphology increased. In this context, our study supports the previous studies and points out that LPO may caused contribute to male reproductive dysfunction by causing sperm morphology abnormalities in diabetic conditions.
DM-induced oxidative stress triggers apoptotic cell death in spermatogonia and spermatocytes [40]. However, molecular mechanism of DM-induced germ cell apoptosis is not fully understood. Nevertheless, there is strong evidence that ROS accumulating with increased oxidative stress activates the intrinsic apoptosis pathway by increasing mitochondrial membrane permeability and promoting cytochrome C release [10]. For example, Zhao et al. showed an increase in pro-apoptotic Bax expression and a decrease in antiapoptotic Bcl-2 expression in diabetic rats' testis [41]. Also, different studies have shown that TUNEL positive germ cell count [42] and caspase (3 and 9) [43] activity are increased in diabetic rats. In herein, we determined an increase in pro-apoptotic Bax and Caspase-3 expressions and a decrease in antiapoptotic Bcl-2 expression in diabetic rats' testis. Furthermore, TUNEL positive germ cells count was high in the diabetic rats’ testis compared to control. In addition to these analyzes, Annexin V method which we use to determine early and late apoptotic sperm cell count showed that the number of late apoptotic cells increases dramatically in diabetic rats.
Today, it is known that drugs used to treat hyperglycemia in DM patients have devastating effects on many other organs, such as the testis. For example, Adaramoye et al. reported that metformin and glibenclamide cause significant reduction in the sperm count and histopathological alteration in testicular tissue via LPO and antioxidant system disruption [44]. Because of these undesirable effects, scientists began to investigate the use of alternative drugs to control the complications of diabetes. One of these drugs is losartan. Clinical and pre-clinical studies have shown that this drug has a delaying effect on DM-related complications such as diabetic cardiomyopathy [45]. On the other hand, there is a large gap in the literature regarding the effects of losartan on germ cell apoptosis associated with DM-induced testicular toxicity. Present study was planned to determine the effect of losartan against DM-induced testicular germ cell apoptosis, oxidative stress, and histopathological change. Results of the present study pointed out that mid-dose losartan treatment may mitigate the testicular tissue degeneration and significantly regulate LPO and SOD activity via restoring the histology/biochemistry of testis. Previous studies have shown that losartan administering to diabetic rats has a protective effect by cleaning LPO products in pancreatic and retinal cells [13, 15]. These reports support to results of this study and result of this study suggest that mid-dose losartan may have an antioxidant effect on diabetic testicular cells such as spermatogonia, spermatocyte and Leydig cells. Besides, the increase in testosterone level in parallel with the decrease in Leydig cell loss indicates that mid-dose losartan may affect hormonal regulation of spermatogenesis.
The immunohistochemical and Real-Time PCR analysis of the study suggested that mid-dose losartan could attenuate apoptotic cell death in diabetic testicular germ cells. The analysis showed that pro-apoptotic Bax and cleaved-Caspase 3 protein and mRNA expression decreased in the diabetic group administering mid-dose losartan, whereas antiapoptotic Bcl-2 protein and mRNA expression increased. In this group, TUNEL (+) testicular germ cell number was found to significantly lower compared to other diabetic groups. In a study with similar results, Yu et al. revealed that Ang-II-induced apoptosis was suppressed in the glomerular podocytes of diabetic rats treated with losartan [14]. Recently, Wang et al. showed that losartan treatment in cavernous nerve injury rat model produced a protective effect by suppressing the expression of pro-apoptotic molecules such as Bax and Caspase 3 in cavernous cells and supporting the expression of antiapoptotic molecules such as Bcl-2 [46]. Furthermore, Bolat et al. reported that losartan administration in the experimental varicocele model reduced TUNEL (+) testicular germ cell count [47]. When the results of our study and the literature are evaluated together, it can be suggested that losartan supports survival of testicular germ cells by suppressing apoptosis. Besides, decrease in the number of late apoptotic cells despite the increase in the number of early apoptotic cells in medium and high dose losartan groups observed in Annexin V tests is another noteworthy finding. This finding supports the argument that moderate dose losartan administration can increase the chances of survival of these cells by suppressing testicular germ cell apoptosis at an early stage.
Further studies are required to analyze the mechanisms underlying this possible antiapoptotic effect of losartan. On the other hand, there are clues that Ang-II inhibition may be one of these mechanisms. On the other hand, there are clues that Ang-II inhibition may be one of these mechanisms. For example, Ang-II activates apoptosis under diabetic conditions and causes damage to many organs, including the testis [48, 49]. Moreover, Ang-II overactivation causes inactivation of Leydig cells and inhibition of steroidogenesis in testes [50]. Therefore, Ang-II inhibition in diabetic conditions may be a promising target in the prevention or treatment of DM-related male reproductive problems.
In conclusion, findings of present study indicated that mid-dose losartan administration may have a therapeutic potential in diabetic testis by reducing the LPO, supporting the antioxidant system and inhibiting the apoptosis. In addition, inhibition of Ang-II provides a candidate approach in the treatment of male fertility problems associated with DM.