Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats

Abstract The purpose of this study was to characterize the testicular profile of Zucker diabetic fatty (ZDF) rats presenting with type 2 diabetes mellitus (DM2) in the absence or presence of obesity. To achieve this, testes were collected from 270-day-old male Wistar (n = 15), ZDF nonobese (n = 15), and ZDF obese rats (n = 16). Changes to the testicular structure were quantified morphometrically, while immunocytochemistry was employed to assess caspase-3 activity. Reactive oxygen species (ROS) production, fluctuations of major antioxidant molecules, and the extent of damage to the proteins and lipids were assessed in tissue lysates. Levels of selected interleukins (ILs) were determined by enzyme-linked immunosorbent assay. The results reveal significant alterations to the testicular structure accompanied by caspase-3 overexpression, particularly in ZDF obese rats. The most notable disruption of the oxidative balance, characterized by ROS overproduction, antioxidant deficiency, protein, and lipid deterioration was recorded in ZDF rats suffering from both DM2 and obesity. Accordingly, the highest concentrations of pro-inflammatory IL-1, IL-6, and IL-18 accompanied by reduced levels of the anti-inflammatory IL-10 were found in testicular tissue collected from ZDF obese rats. This study highlights the vulnerability of male gonads to pathophysiological changes caused by hyperglycemia, which are further exacerbated by excessive adipose tissue.


Introduction
Diabetes mellitus (DM) comprises a group of metabolic ailments characterized by hyperglycemia arising from alterations in the secretion and/or activity of insulin. The resulting chronic high blood sugar levels may contribute to the dysfunction of numerous organs, particularly the kidneys, eyes, nerves, heart, and blood vessels [1,2]. The underlying cause for DM-inflicted damage to the metabolism of carbohydrates, lipids, and proteins lies in an insufficient action of insulin on target tissues. This phenomenon is triggered by either inadequate insulin secretion or a decreased response of cellular targets to insulin at one or more points of the endocrine network. A decreased insulin secretion and activity often coexist in one patient; hence, it may be difficult to determine which of these abnormalities is directly responsible for the development of hyperglycemia [2]. DM may be divided into three types: DM type 1 (DM1), DM type 2 (DM2), and DM type 3 (DM3)also known as gestational DM. DM1 is triggered by autoimmune destruction of pancreatic beta cells (β-cells) and is generally diagnosed in children or adolescents [1,2].
According to Reed et al. [3], DM2 is nowadays the most prevailing metabolic disease, affecting more than 450 million people worldwide. DM2 pathogenesis is characterized by abnormalities in glucose and lipid metabolism, including insufficient insulin secretion by the β-cells and insulin resistance. Target cells are then unable to absorb glucose, leading to the occurrence of hyperglycemia [1,3]. Chronically elevated concentrations of blood sugar may subsequently promote the accumulation of polyols, oxidative insults to healthy tissues, chronic inflammation, and cell death through apoptosis or necrosis [3][4][5]. While the exact etiology of DM2 is still not fully understood, it is known that being obese increases the risk of developing the disease. In fact, the recent research suggests that obese people are up to 80 times more likely to present with DM2 than those with a normal body mass index [6]. A frequent phenomenon interlinking DM with obesity is the metabolic syndrome, which refers to a complex of health issues including insulin resistance, hyperinsulinemia, hyperglycemia, hypertension, and hypercholesterolemia [6,7].
Male gonads may be characterized by a unique architecture, organization, and function. A continuously ongoing process of spermatogenesis presents with high energy requirements and an exceptional metabolic activity of male germ cells, which on the other hand renders the testes to be a highly sensitive barometer to any imbalance of the internal milieu or external stimuli. As reviewed by Condorelli et al. [8], DM could have detrimental effects on male fertility, most notably on the semen quality, translated into decreased sperm motion, excessive DNA fragmentation, and abnormal seminal plasma characteristics. Similar to DM, obesity has been acknowledged as a factor substantially contributing to suboptimal male reproductive performance. Fertility among obese subjects may be affected by sexual dysfunction, endocrinopathy, aromatization activity, thermal stress, as well as inflammatory and obstructive elements of testicular and epididymal pathology [9]. While DM2 and obesity share a mutual relationship, their precise mechanism of action on the male reproductive system needs further understanding.
Animal models play a pivotal role in monitoring and understanding DM pathogenesis due to a combination of their genetic and functional characteristics. Nevertheless, most studies take advantage of rodents subjected to streptozotocin (STZ) that causes pancreatic β-cell destruction and is widely used to induce symptoms consistent with DM1. In the meantime, metabolic studies by and large employ a high-fat diet-induced obesity in animals, which may or may not develop DM2 [10]. To assess a shared impact of obesity and DM2, Zucker diabetic fatty (ZDF) rats seem to act as an optimal model. ZDF rats present with a mutation in the leptin receptor gene, which causes leptin unable to bind to the saturation center in the brain. The animal suffers from consistent hunger and develops obesity when fed, which is subsequently followed by the occurrence of DM2. The animals are characterized by progressive β-cell dysfunction, glucose intolerance, severe insulin resistance, and hyperlipidemia and become diabetic at 8 weeks of age when fed a diet containing at least 6.5% fat [11]. This profile mimics a progressive loss of glucose-stimulated insulin secretion in humans suffering from DM2, and hence, ZDF rats represent the best laboratory model to study human DM2, its pathophysiology, and the effects of potential therapeutic options [11][12][13].
Despite an array of studies employing male ZDF rats ref. [11], only a few reports have strived to assess their reproductive characteristics. Evidence gathered from these studies indicates a marked testicular atrophy accompanied by the detachment and disorganization of germ cells [14][15][16][17]. Moreover, a global transcriptomic analysis revealed changes in the genes involved in the lipid metabolism [18], which may lead to a diminished fertility in ZDF males. Nevertheless, very little is known about the involvement of oxidative stress and inflammation in the reproductive dysfunction of ZDF rats. As such, our aim is to provide a comprehensive description of the histological, oxidative, and immunological profile of the testicular tissue collected from ZDF rats diagnosed with DM2 in the presence or absence of obesity.

Animals
This study employed 46 adult (270 days) male rats, which were obtained from the Institute of Experimental Pharmacology (Slovak Academy of Sciences, Dobrá Voda, Slovakia) and kept in plastic cages at 24 ± 1°C and a 12 h light/12 h dark photoperiod. All animals were provided with drinking water ad libitum [19,20].
The rats were divided into three groups: the control group included Wistar rats (n = 15), the first experimental group comprised ZDF (fa/fa) nonobese rats (n = 15; ZDF nonobese (ZN)), and the second one consisted of ZDF (fa/fa) obese rats (n = 16; ZDF obese (ZO)). The control and ZN groups were fed under a controlled regime, while the ZO group had unrestricted access to the chow (Purina Rodent LabDiet 5008, IPS Product Supplies, UK) with a fat content of 6.50%. Fasting blood glucose levels were monitored using a FreeStyle Optium Neo Glucose and Ketone Monitoring System (Abbott Diabetes Care Ltd., UK) with a measurable extent of 1.1-27.8 mmol/L. One blood drop was obtained from the tail vein in the morning between 6:30 and 8:00 A.M. every 2 weeks. Diabetes was acknowledged when the blood glucose level was equal to or higher than 17 mmol/L [19]. By week 8, all ZN and ZO rats had developed hyperglycemia that remained persistent until animal sacrifice.
The final glycemic measurement ( Table 1) was performed shortly before animal sacrifice by sevoflurane anesthesia and decapitation. Testes were immediately excised from the scrotum, dissected from the epididymis, and weighted. The right testes were processed for histological analyses, while the left testes were used for lysis and subsequent biochemical assays. A resume of the experimental approach is outlined in Figure 1.

Immunocytochemistry
For the immunocytochemical assessment of caspase-3 activity, testicular tissue was fixed in 10% formol, dehydrated in ethanol series, embedded with paraffin, and sectioned into 5 µm sections. The resulting sections were deparaffinized in xylene (Sigma-Aldrich) and rehydrated in a graded series of ethanol. Subsequently, the sections were treated with 10 mM citrate buffer (pH 6.0) twice for 5 min each and cooled down. Following washing three times in phosphate buffer (PBS; Sigma-Aldrich), endogenous peroxidases were deactivated by 3% hydrogen peroxide (H 2 O 2 ; Sigma-Aldrich) for 20 min, and the sections were washed again three times with PBS. For the blocking procedure, the samples were treated with Animal-Free Blocking Solution (Cell Signaling Technology, Danvers, MA, USA) for 10 min. Subsequently, the samples were incubated at 4°C with the rabbit Caspase 3 primary antibody (#9662, 1:1,000; Cell Signaling Technology) overnight. After washing with PBS, the sections were incubated with an antirabbit, horseradish peroxidase-linked secondary antibody (#7074, 1:1,000; Cell Signaling Technology), washed again in PBS, and visualized with diaminobenzidine (10%; Roche Diagnostics Corporation, IN, USA). The sections were finally mounted with the Permount mounting medium (Fisher Scientific, Hampton, NH, USA) on glass slides. Photographs were taken with the Leica EC3 optical microscope at a primary magnification of 10× and 20× (Leica Camera AG; Wetzlar, Germany) [22,23]. Assessment of the immunocytochemical reaction was performed using the H-SCORE system based on a semiquantitative analysis distinguishing among the following categories: 0 (no staining), 1+ (weak/faint yellow staining), 2+ (moderate/brownish yellow staining), and 3+ (intense/ brown staining). The calculation of the H-SCORE followed the formula previously used by Ayan et al. [22]. Five randomly selected areas per each slide were assessed under a light microscope (10× and 20× primary magnification).

Preparation of tissue lysates
For the biochemical assays, testicular tissue samples of approximately 50 mg were pretreated with RIPA buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich) and subsequently sonicated on the ice at 28 kHz for 30 s. Following centrifugation (11,828×g, 4°C, 15 min), the lysates were collected and stored at −80°C [24].
Before analyses of the oxidative and immunological profile, protein concentration was quantified in each lysate, which was necessary for a posterior data normalization. Each lysate was processed with the total protein commercial kit based on the Biuret method (DiaSys Diagnostic Systems, Holzheim, Germany), and the total amount of proteins was assessed with the help of the Rx Monza semi-automatic analyzer (Randox Laboratories Ltd., Crumlin, UK) [24,25].
Protein oxidation expressed through the concentration of protein carbonyls was performed with the 2,4dinitrophenylhydrazine (DNPH) method introduced by Weber et al. [27]. Each lysate was prediluted with distilled water to contain 1 mg protein and pretreated with 1 mL trichloroacetic acid (TCA; 20% w/v; Sigma-Aldrich). Subsequently, 1 mL of each lysate was incubated with 1 mL DNPH (10 mM in 2 N HCl; Sigma-Aldrich) at 37°C for 1 h. Then, 1 mL TCA was added to the mixture, which was then centrifuged (300×g, 15 min). The resulting pellets were washed three times with 1 mL of ethanol/ethyl acetate (1/1; v/v; Sigma-Aldrich) and resuspended in 1 mL 6 mol/L guanidine hydrochloride (Sigma-Aldrich) before absorbance measurement at 360 nm with the Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The amount of protein carbonyls (PCs) was calculated using the molar absorption coefficient of 22,000 1/M.cm and is expressed as nmol PC/mg protein [24].
Malondialdehyde (MDA) as a marker of lipid peroxidation (LPO) was quantified with the thiobarbituric acid reactive substances assay, modified for a 96-well plate. Each lysate was pretreated to 5% sodium dodecyl sulfate (Sigma-Aldrich) and subjected to 0.53% thiobarbituric acid (Sigma-Aldrich) dissolved in 20% acetic acid (Centralchem, Bratislava, Slovakia). The samples were boiled at 100°C for 1 h, then cooled down on the ice for 10 min, and centrifuged at 1,750×g for 10 min. The obtained supernatant (150 µL) was transferred to a 96-well plate and subjected to absorbance measurement at 540 nm using the Glomax Multi + combined spectro-fluoro-luminometer [24,25]. The results are expressed as µmol MDA/g protein.
Superoxide dismutase (SOD) activity was quantified with the help of the RANSOD commercial kit (Randox Laboratories, Crumlin, UK), while the RANSEL commercial assay (Randox Laboratories, Crumlin, UK) was used for the assessment of glutathione (GSH) peroxidase (GPx) activity. The lysates were processed according to the instructions of the manufacturer, and activities of both antioxidant enzymes were measured with the Rx Monza semi-automatic analyzer. The results are expressed as IU/g protein [24,26].
To quantify the activity of catalase (CAT), we followed the protocol by Beers and Sizer [28]. Each sample was diluted with 0.05 mol/L PBS (pH 7.0) and subsequently exposed to 0.059 mol/L H 2 O 2 (30%) in 0.05 mol/L PBS. The decrease of H 2 O 2 was tracked at 240 nm using the Cary 60 UV-Vis spectrophotometer, and CAT activity was calculated from the initial linear portion of the rate curve.
The obtained values are expressed as IU/mg protein [24,25].

Immunological analysis
Levels of interleukin (IL)-1, IL-6, IL-10, and IL-18 were evaluated using commercially available ELISA kits suitable for rat tissue lysates (#RAB0278, #RAB0312, #RAB0247, and #RAB1147, respectively; Sigma-Aldrich). All assays followed a double-sandwich protocol and were carried out according to the instructions of the manufacturer. IL concentrations were determined with the Glomax plate spectrophotometer (Promega Corporation, Madison, WI, USA) at 450 nm and are expressed as pg/mg protein.

Statistical analysis
Statistical analysis was carried out using the GraphPad Prism program (version 8.4.4 for Mac; GraphPad Software Incorporated, La Jolla, CA, USA). The results are expressed as mean ± standard deviation. First, the data were analyzed using the Shapiro-Wilk normality test taking a normal (Gaussian) distribution into consideration. All data sets passed the test with nonsignificant results at the alpha level of 0.05. Differences between the groups were analyzed using one-way analysis of variance followed by the Tukey multiple comparison test, designed to compare the means of three or more independent samples simultaneously. P-values <0.05 were considered statistically significant.

Testicular histology and morphometry
Results obtained from the histological and morphometrical analyses are presented in Table 1. The testicular weight was decreased in both experimental groups in comparison with the control. A significant reduction of the testicular weight was recorded in the ZN group when compared with the control (P < 0.05). We also observed that the testicular weight in the ZO group was higher in comparison with that in the ZN group.
Testicular tissue of the control group presented with normal, closely associated seminiferous tubules and intact basal membranes. The spermatogenic line was undisrupted and contained all the layers of spermatogenic cells. The lumen was filled with spermatozoa. No obvious disruptions to the structure or infiltrations to the interstitial space were detected (Figure 2a and b). Testicular structures in the ZN group exhibited irregular basal membranes in numerous seminiferous tubules with disruptions in the seminal epithelium and the spermatogenic series. Atrophic changes were observed in several specimens (Figure 2c and d). Many tubules in the ZO group were structurally damaged with the seminal epithelium being detached from the basal lamina. In numerous cases, the spermatogenic cycle was disrupted with signs of necrosis and denuded spermatogenic cells. The lumen of several seminiferous tubules contained cell debris (Figure 2e and f).
Microscopic observations were furthermore supported by the semi-quantitative analysis of photomicrographs ( Table 1). The highest relative volume of the seminal epithelium was found in the control group. Both experimental groups presented a significant decrease in the relative volume of the epithelium in comparison with the control (P < 0.05). On the contrary, the highest proportion of the luminar relative volume was found in the ZO group, which was significantly higher when compared with the control (P < 0.05).
Assessment of the morphometric characteristics of the seminiferous tubules revealed a continuous decline of the tubular and luminar diameter proportionately to the severity of the metabolic disease. The lowest diameter of the seminiferous tubules as well as the luminar diameter were found in the ZO group, which was significantly different when compared with the control (P < 0.05) and the ZN group (P < 0.05). A similar phenomenon was observed in the case of the epithelial height, which was significantly lower in the ZN group as well as in the ZO group in comparison to the control (P < 0.05; Table 1).

Immunocytochemistry
Caspase-3 expression was detected particularly within the primary and secondary spermatocytes in all three groups with different intensities. The control group exhibited a weak caspase-3 signal (Figure 3a and b), whereas a moderate expression of the proapoptotic enzyme was observed in the ZN group (Figure 3b and d) with a significantly higher H-SCORE in comparison with the control (P < 0.05; Figure 3g). The highest caspase-3 activity was detected in the ZO group with a strong immunocytochemical expression pattern in the whole germline including Sertoli and Leydig cells (Figure 3e and f). The resulting H-SCORE was significantly higher when compared with the control (P < 0.05; Figure 3g) as well as the ZN group (P < 0.05; Figure 3g).

Oxidative profile
The chemiluminescent analysis revealed a significant increase of ROS in the ZN group in comparison to the control (P < 0.05). This process was further aggravated by DM2 and obesity as reflected by significantly higher concentrations of ROS in the ZO group when compared to the control (P < 0.05) and the ZN group (P < 0.05; Figure 4a), indicating a higher risk for the development of testicular oxidative stress in the animals suffering from chronic hyperglycemia and obesity. A disturbance of the oxidative milieu was corroborated by a significant decline of TAC in both experimental groups when compared with the control (P < 0.05 in the case of ZN; P < 0.01 with respect to ZO; Figure 4b). The lowest antioxidant strength of the testicular tissue was observed in the ZO group, which was significantly diminished even in comparison with the ZN group (P < 0.05).
As unraveled by the DNPH assay, a significantly increased occurrence of protein carbonyls was observed in both experimental groups when compared with the control (P < 0.05; Figure 4c). At the same time, DM2 had a profound impact on the extent of testicular LPO, as evidenced by a significant increase in MDA levels in the ZN group when compared to the control (P < 0.05). Obesity aggravated the intensity of LPO since the amounts of MDA were significantly higher in the ZO group in comparison with both the control and the ZN group (P < 0.05; Figure 4d).
A deeper look at the principal components of the antioxidant system of male gonads revealed that SOD activity was declining correspondently to the severity of the ailment. A significantly lower SOD activity was detected in the ZN group in comparison with the control (P < 0.05). The lowest activity of the enzyme was observed in the ZO group, which was significantly different in comparison to both the control and the ZN group (P < 0.05, Figure 5a). CAT seemed to be impacted by both metabolic diseases even more, since a significantly lower activity of this antioxidant enzyme was recorded in both experimental groups as opposed to the control (P < 0.05). A significantly different CAT activity was also observed when comparing the ZN and ZO groups (P < 0.05; Figure 5b). GPx responded to DM2 or DM2 and obesity in a similar manner as did SOD and CAT. A significantly lower GPx activity was recorded in the ZN group in comparison with the control (P < 0.05). Nevertheless, the lowest activity of the enzyme was detected in the ZO experimental group, which was significantly different in comparison with both the control and the ZN group (P < 0.05; Figure 5c).
A decreased activity in the prime enzyme of the GSH cycle was well reflected in the dynamics of reduced GSH among the groups distributed in this study. While a decline in the GSH concentration was observed in the ZN group (P < 0.05), the lowest amounts of this nonenzymatic antioxidant   were detected in the ZO group with significant differences in comparison to both the control and the ZN group (P < 0.05; Figure 5d).

Immunological profile
As shown in Figure 6a, IL-1 concentration increased in both experimental groups; however, significant differences were observed only between the control and the ZO group (P < 0.05). A similar trend was recorded in the case of IL-6; however, significant differences were noted in both experimental groups when compared to the control group (P < 0.05; Figure 6b). Inversely, a continuous decrease of IL-10 was observed in the experimental groups. The lowest levels were recorded in the ZO group, which were significantly different in comparison with the control (P < 0.05, Figure 6c). Nevertheless, IL-18 revealed an increasing trend corresponding to the health complications of the animals with the highest concentrations detected in the ZO group (P < 0.05 in comparison with the control; Figure 6d).

Discussion
It is well known that diabetes plays a substantial role in testicular pathophysiology through inducing tubular atrophy, damage to the spermatogenic line, and alterations to the blood-testis barrier, which together with chronic inflammation and oxidative stress may contribute to a reduced sperm function [7,30].
A close relationship between DM2 and obesity has been known for decades. Obesity per se has a negative impact on male reproduction via endocrine alterations, abnormal sperm function, or changes to the molecular characteristics of the reproductive system. Evidence suggests that impaired spermatogenesis and sexual dysfunction are the primary causes responsible for poor fertility in obese subjects [reviewed by ref. [9]]. Nevertheless, little is known about the impact of the metabolic syndrome encompassing both obesity and DM2 on the testicular architecture and microenvironment. As such, we strived to characterize the structural, oxidative, and immunological properties of testicular tissue collected from ZDF rats suffering from DM2 alone or from DM2 and obesity. Testicular weight measurements in our study corroborate observations from previous investigations [14,17,31], suggesting that a lower weight of male gonads may have been caused by an ongoing hyperglycemia that leads to the loss of muscle and adipose tissue due to exhaustion of available sources of energy. Conversely, animals suffering from DM2 and obesity displayed higher testicular weight in comparison to ZDF lean rats, most likely due to the presence of excess adipose tissue that seems to compensate for the loss of nourishment for the reproductive cells [32].
Testicular dysfunction is a typical side effect of DM-associated male subfertility. In accordance with our morphometric analysis, previous reports found that the induction of diabetes led to a reduction of the germ cell count that was accompanied by alterations to the spermatogenic series [14,15,33,34]. Correspondingly, DM has been frequently correlated with testicular atrophy and a decline in the proportions of seminiferous tubules, which indicates failure of proper spermatogenesis [16,34,35]. Even though the exact molecular mechanism involved in DMassociated testicular disintegration is not fully understood, it is believed that hyperglycemia or poor glycemic control may be critically involved in this process [36]. Chronic testicular hyperglycemia may lead to impaired communication of the hypothalamic-pituitary-gonadal axis, a disrupted sympathetic innervation and cellular signaling, oxidative stress, increased DNA damage, inhibition of the mitochondrial metabolism, and increased endoplasmic reticulum stress [36][37][38]. Moreover, Maresch et al. [38] suggested that persistently high glucose levels may disrupt prime glucose metabolism pathways, leading to an accumulation of the respective end products within the reproductive tract.
A more severe damage to the testicular tissue was observed in the case of rats suffering from both DM2 and obesity. In accordance with our morphometric data, a meta-analysis by Zhong et al. [39] uncovered reports indicating histopathological alterations in the seminiferous epithelium, accompanied by the presence of cellular debris and germ cell detachment from the basal lamina in testicular tissue from obese subjects. Sertoli cell vacuolization, scattered intraluminal giant cells, and a significant decline of the germinal epithelium thickness were reported as well. On the other hand, Vendramini et al. [14] and Mansour et al. [16] did not observe significant changes in the morphometric properties among ZDF lean and obese rats. This discrepancy may be explained by the age of the animals used in the experiments. While both studies employed pubertal or young adults, we chose to study rats of approximately 9 months of age. The decision to employ older rats was based on selecting a timeframe when complications arising from DM2 were well visible, while the animals were still within their peak reproductive performance [20]. Moreover, as proposed by Salama et al. [17], changes to male reproductive structures caused by DM2 are age dependent and are more pronounced as time progresses.
As previously discussed, several reasons may lay behind a fertility decline in obesity. The endocrine activity of adipocytes may interfere with the hypothalamic-pituitarytesticular axis, affecting the levels of follicle-stimulating hormone, luteinizing hormone, and testosterone [40]. Excessive adipose tissue enclosing the testes may furthermore increase the scrotal temperature leading to a lower testicular weight and sperm quality [41]. Obesity has also been associated with ROS overproduction and DNA fragmentation of the male germ cell line [42]. Finally, global transcriptomic and metabolomic studies uncovered severe alterations in the metabolism of lipids and fatty acids in the testes of ZDF rats, which are essential for normal spermatogenesis and steroidogenesis [18,43].
Our immunocytochemical data suggest that DM2 and/or obesity promote testicular apoptosis in a manner comparable to earlier reports [44,45]. According to Zha et al. [46] and Zhao et al. [47], chronic hyperglycemia alters the testicular proapoptotic Bax/antiapoptotic Bcl-2 ratio. Subsequent upregulation of p53, caspase-8, and caspase-9, indicative of apoptotic signaling, triggers caspase-3. Caspase-3 activation as a major executor of apoptosis recorded in this study was furthermore validated by Nna et al. [48], who observed an upregulation of both mRNA and protein levels of caspase-3 followed by an increased testicular cell apoptosis. Correspondingly, He et al. [45] recorded a notable caspase-3 immunocytochemical signal in spermatogonia, spermatocytes, and Sertoli cells of rats suffering from severe hyperglycemia induced by STZ and high-fat diet.
Oxidative stress, defined as a shift of the prooxidant-antioxidant balance toward ROS production, has been recognized as a key hallmark of hyperglycemia-induced testicular damage [38,49]. Diabetes may be accompanied by ROS overgeneration during the mitochondrial glucose oxidation, which results from high blood sugar levels. Mitochondrial metabolism is sensitive to excessive ROS, and it has been previously established that hyperglycemia represses mitochondrial respiration [5,49]. Excessive amounts of ROS as observed in our study will be subsequently released into the cytoplasm of cells assembling the seminiferous epithelium, outweighing the antioxidant defense system [48,49], and disrupting the spermatogenic process [50]. Testicular germ cells are highly susceptible to oxidative insults because the backbone of their plasma membranes comprises polyunsaturated fatty acids prone to oxidation [51], which may explain a significant increase in LPO recorded in our experimental groups. Furthermore, an increased oxidative pressure on the germ cells may favor an upregulation of caspase-3 and subsequent cell death [49,52,53], which correlates with our immunocytochemical analysis.
Oxidative damage may be even more aggravated if diabetes is associated with obesity, as revealed by our assessment of the testicular oxidative profile. Obesity is defined as an inflammatory condition that is accompanied by a higher metabolic rate and subsequent release of ROS into testicular structures. Pro-inflammatory cytokines may also cause severe damage to the seminiferous epithelium by triggering ROS overgeneration [54,55]. Such activation of the cytokine network will further attract infiltrating phagocytes that are inherently predisposed to release ROS during oxidative outbursts [56].
A disruption of the testicular antioxidant power as observed in the ZN and more so in the ZO group indicates a shift in the oxidative milieu, which may be orchestrated by increased intracellular ROS levels and a higher incidence of oxidative damage to the cells [49,50,57]. Furthermore, this imbalance may be accompanied by a downregulation of critical components of the innate antioxidant capacity, which is consistent with previous studies [44,49,58,59]. The infectivity of SOD to catalyze the dismutation of superoxide to H 2 O 2 , followed by a decreased H 2 O 2 decomposition to water by CAT and/or the GSH cycle, will lead to its accumulation and subsequent damage to proteins and lipids [60]. As such, our data strongly indicate that the SOD-CAT-GPx antioxidant triangle was significantly impaired in the testes of rats suffering from hyperglycemia, which may have resulted in an increased H 2 O 2 infiltration to male reproductive tissues.
Diabetes, obesity, and the resulting metabolic syndrome represent a cluster of conditions that may trigger the release of pro-inflammatory biomarkers, which are predictive of insulin resistance [61][62][63]. As suggested by previous studies, chronic hyperglycemia is accompanied by the activation of two major inflammatory pathways, specifically the stress-activated Jun N-terminal kinases and the transcription factor nuclear factor-kappa B pathway [64,65]. This inflammatory state accelerated by the release of proinflammatory cytokines may be further promoted by the activity of adipokines since these may stimulate additional independent inflammatory responses in obesity [4,66,67]. Among a vast array of molecules considered to act as adipokines, tumor necrosis factor alpha, leptin, adiponectin, ILs, monocyte chemoattractant protein, resistin, and chemokines play major roles in mediating inflammation and causing further disruptions to the glucose homeostasis [55,64,67,68].
In this study, we assessed four major ILs that have been implied in male reproduction. As expected, IL-1 and IL-6 were increasing parallel to the severity of DM and/or obesity in the experimental animals. Accordingly, increased levels of IL-1 have been recorded during a systemic or local inflammation and directly affected testicular steroidogenesis [69]. Similarly, high amounts of IL-6 have been often observed during inflammatory stress that was accompanied by a considerable reduction of testosterone levels [68,70]. In the male gonads, pro-inflammatory cytokines including IL-1 and IL-6 may affect the expression and assembly of the junctional and cytoskeletal proteins, thereby inducing openings of the cell junctions between the adjacent Sertoli and epithelial cells, leading to alterations in the niche of seminiferous epithelium that is essential for spermatogenesis [71,72].
IL-18 is a less known molecule, which may increase particularly during autoimmune pathologies [73] and contribute to the formation of antisperm antibodies that are a common reason for male infertility during systemic inflammation [74]. Elevated concentrations of IL-18 in this study may provide a parallel to caspase-3 expression patterns since it has been suggested that IL-18 is involved in the control of testicular cell proliferation and apoptosis. According to several reports, high IL-18 activity could also inhibit spermatogenesis in sexually mature individuals through enhanced oxygen metabolism [75]. On the other hand, IL-10 represents a major anti-inflammatory cytokine, which was found to be downregulated in patients with impaired fertility. A similar relationship was observed in men with testicular inflammation [76,77], suggesting that IL-10 may inhibit the recognition of antigens by T lymphocytes [78]. In our case, a continuous decrease in this cytokine was observed in both experimental groups, indicating that besides oxidative imbalance, DM2 and/or obesity may disrupt the cytokine network favoring a pro-inflammatory state. Such phenomenon may then cause damage to the testicular tissue and decrease immune protection of male reproductive cells.
Summarizing our experimental data and supporting evidence, we may conclude that the metabolic syndrome associated with DM2 and/or obesity leads to serious alterations in the testicular oxidative profile caused by ROS overproduction, with a concomitant failure of inherent antioxidant mechanisms as well as structural and/or functional damage of proteins and lipids involved in the process of spermatogenesis. Furthermore, chronic inflammation triggered by hyperglycemia, adipokines, and oxidative stress may lead to an imbalance in the immune network, causing an additional risk to the testicular structure and function. All aforementioned phenomena may ultimately result in testicular disintegration and increased germ cell apoptosis, which may contribute to male reproductive dysfunction as a consequence of DM2.

Conclusion
Our results suggest that the testicular architecture is highly susceptible to pathophysiological changes associated with DM2. Oxidative stress and inflammation may be involved in the progression of testicular dysfunction, which may lead to male subfertility in diabetic and/or obese patients. Finally, we may encourage to use ZDF rats to study therapeutic options in the prevention and/or management of male infertility associated with the progression of DM2.