Epidermal growth factor (EGF) is a 53-amino acid polypeptide that is a potent mitogen in various tissues . It was first discovered by Cohn in 1962 as the principle substance in extracts of submandibular glands of male mouse. It is synthesized by the granular convoluted tubule (GCT) of rodent submandibular glands . In man, EGF has been localized either in the duct cells  or in serous cells . It is important in many physiological processes as spermatogenesis, completion of normal pregnancy, mammary gland development and wound healing . EGF is the active ligand for EGF receptor reported in germ cells, and proper EGF expression is important for completion of spermatogenesis. Removal of the salivary gland in rodents, which reduces circulating EGF, reduces spermatogenesis . EGF deficiency contributes to the pathology of many disease states. It was proved that in diabetic mice, the levels of EGF, its messenger RNA in the submandibular glands and the circulating level were greatly reduced . Oxidative stress is currently suggested as the mechanism that is involved underlying diabetes and diabetic complications . Sildenafil is a selective cyclic GMP-phosphodiesterase inhibitor used in the treatment of sexual dysfunctions. The active compound (sildenafil citrate) is present in Viagra. It has stimulated submandibular secretion of protein, EGF and flow rate of saliva in normal rats by quantitative immunoassay techniques . Treatment of diabetic rats by phosphodiesterase inhibitors restored the oxidative stress markers near normal levels in the order of milrinone>sildenafil>theophylline . Many studies have discussed the role of antioxidants in the management of diabetes and its complications, but relatively little attention has been paid to the effect of sildenafil (Viagra) on EGF production by the submandibular gland in cases of diabetes. This research aimed to study the influence of sildenafil (Viagra) on EGF production by the submandibular glands in male diabetic rats, and the expression of its receptors on target organs as the testis in an attempt to evaluate sildenafil therapeutic role in diabetes-induced EGF deficiency. In addition, B cells of islets of Langerhans will be assessed.
Materials and methods
Animals and experimental design
Sixty male Wister-albino adult rats, weighing 150–200 g, were used in this study. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Assiut University, Faculty of Medicine. The groups were put in separate cages with free access to food and water, and were weighed for monitoring their food and water consumption. They were divided into three main groups, each included 20 rats: group 1 (control group): received saline 10 ml/kg intraperitoneally daily for a month. Group 2 (diabetic group): received alloxan once at day 1 of the experiment at a dose of 150 mg/kg by intraperitoneal injection; then after confirmed for diabetes (within 72 h) were killed a month later. Group 3 (treated diabetic group): received alloxan once at day 1 of the experiment; then after confirmation of diabetes (72 h later) treated with sildenafil 1 mg/kg orally by a gastric tube daily for 1 month. All rats were killed by decapitation, 1 month after diabetes confirmation.
An experimental model of type II diabetes mellitus was set up in rats by single alloxan injection, which is a classical diabetogenic agent. Oral treatment with sildenafil in a single daily dose for 1 month was administered to mimic human therapeutic intake. Detailed histological, immunohistochemical and ultrastructural study of the submandibular glands and pancreatic islets of Langerhans, respectively, was achieved. The presence of EGF receptor was investigated in the testis.
Drugs, dosage and administration
Alloxan monohydrate dissolved in normal saline was used for induction of diabetes at a single dose of 150 mg/kg by intraperitoneal injection . Sildenafil in the form of commercial tablets (Viagra), a product of Pfizer Company (Pfizer Egypt, SAE, Cairo), was used. Each tablet contains 100 mg of the active principle (sildenafil citrate). To prepare suspension of the drug, one tablet was grinded and was then suspended in 100 ml of distilled water, so that each 1 ml of the suspension contained 1 mg of the active principle sildenafil citrate. The dose chosen for this study was 1 mg/kg , and was administered by a polyethylene gastric tube.
After alloxan injection, diabetes was confirmed by measuring daily blood sugar from the tail vein with On Check sticks. Hyperglycaemic rats (200–300 mg/dl)  were included in the study.
Histological and immunohistochemical studies
The submandibular glands and the testes were removed and immersed in 10% buffered neutral formalin for 48 h, washed in running tap water, dehydrated and then paraffin embedded. Paraffin sections (4 μm thick) were mounted on positively charged glass slides with polylysine, and were stained with EGF and EGF receptor immunohistochemistry as follows:
Immunohistochemistry was performed according to manufacturer's protocol. Tissue sections were deparaffinized, rehydrated in graded alcohol and transferred to phosphate-buffered saline (PBS; pH 7.6). The slides were rinsed twice with PBS, and then endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in methanol for 5 min.
After three times wash with PBS, antigen retrieval for EGF receptor slides was performed by redigestion in protease enzyme for 5 min. No retrieval was required for EGF slides. The latter slides were incubated for 1 h at room temperature with primary antibody for EGF, mouse monoclonal antibody (EGF-10: sc-57088 Santa Cruz Biotechnology Inc., California, USA) at a dilution of 1 : 50. For EGF receptors, the slides were incubated for 18 h (overnight) at 4°C with primary antibody for EGF receptor, mouse monoclonal antibody (Ab-15, Thermo Scientific, Fremont, California, USA) at a dilution of 1 : 50. The slides were then rinsed three times with PBS, and were incubated for 10 min with the biotinylated goat antipolyvalent (Thermo Scientific) at room temperature. After further rinsing with PBS, the slides were incubated for 10 min with Streptavidin peroxidase (Thermo Scientific) at room temperature. The slides were again washed three times with PBS, and diaminobenzidine was applied for 5 min at room temperature. Finally, the slides were rinsed in distilled water, counterstained with Mayer's haematoxylin, dehydrated and mounted. Positive control sections for EGF and EGF receptor antibody were from oral squamous cell carcinoma . Specificity of staining was checked on negative control slides by omitting the primary antibody.
For EGF, a distinct brown cytoplasmic staining was scored as positive. For EGF receptor, membranous and cytoplasmic brown staining was remarkable. Qualitative estimation of the relative immunostaining of EGF-positive cells was graded as −, undetectable; +, weak; 2+, strong; 3–4+, very strong or intense .
Histomorphometric study was carried out to
- (1) Count GCTs in submandibular glands in five semithin sections of the submandibular gland of each group at a magnification of ×100. Three nonoverlapping fields were chosen in each section.
- (2) Count the immunolabelled cells for EGF in the duct system of submandibular glands. Twelve nonoverlapping fields from randomly chosen 10 sections for each group were captured at a ×400 magnification with the aid of a digital camera placed on a Leica microscope. Counting was done using an image analyzing system software (Leica Q 500 MCO) in the Histology department, Faculty of Medicine, Assuit University.
Data were expressed as mean±standard deviation. Comparison between groups was done using analysis of variance followed by a posthoc (least significance difference) test for pairwise comparison between groups using SPSS program version 16 (SPSS INC., Chicago, Illionois, USA). Significance was accepted when the P value was less than 0.05.
Electron microscopic study
Specimens from the pancreatic tails and submandibular salivary glands were trimmed and fixed in 2.5% glutaraldehyde for 24 h. Then, they were postfixed in 1% osmium tetroxide and embedded in epoxy resin for 48 h. Semithin sections (1 μm) were prepared and stained by toluidine blue. Ultrathin sections (0.07 μm) of only pancreatic islets were cut and stained with uranyl acetate and lead citrate . They were subsequently examined and photographed under a JEOL transmission electron microscope in Assiut University.
Submandibular salivary glands
Control group: examination of toluidine blue-stained semithin sections revealed the normal histological structure of the gland comprising the secretory acini and the duct system (Fig. 1a). The GCTs were found in discrete groups in close relationship with the striated ducts (Fig. 1a). The granular cells lining the tubules were characterized by vesicular basal nuclei and abundant deeply stained secretory granules that filled approximately the apical two thirds of the cell. The granules varied greatly in size and shape (Fig. 1b).
EGF intense immunoreactivity in the duct cells was recognized as dark brown color (Fig. 2). EGF receptor intense immunoreactivity was present in the cell membrane and cytoplasm of seminiferous tubule cells and interstitial cells (Fig. 3).
Diabetic group: semithin sections showed regression of the GCTs (Fig. 4). The granular cells showed vacuolation compressing the nuclei (Fig. 5a). Some granular cell nuclei appeared darkly stained (Fig. 5b). The amount of secretory granules was reduced variably, even disappeared (Fig. 5b).
Immunohistochemically, EGF was only weekly expressed through the duct cells, which were hardly identifiable (Fig. 6).
Weak reaction for EGF receptor in the seminiferous tubules of the testis and interstitial cells was observed (Fig. 7).
Sildenafil-treated diabetic group: the GCTs exhibited a pattern relatively similar to those of the control. They were uniformly distributed among the secretory acini (Fig. 8a). The lining granular cells showed rounded vesicular nuclei and plentiful pleomorphic secretory granules (Fig. 8b).
In sections stained for EGF by immunohistochemistry, the duct system showed strong immunoreactivity of its lining cells compared with the diabetic group (Fig. 9).
EGF receptor positive immunoreactivity in the testis was relatively stronger than that observed in the diabetic group (Fig. 10).
Islets of Langerhans
Control group: semithin sections of the pancreas stained with toluidine blue showed the islets of Langerhans as well defined groups of small cells with pale granular cytoplasm and vesicular nuclei, interspersed with blood capillaries among strongly stained exocrine pancreatic acini (Fig. 11a).
Electron microscopic examination of control B cells revealed characteristic electron-dense core granules in the cytoplasm surrounded by an electron-lucent halo. Euchromatic rounded nucleus and other organelles including rough endoplasmic reticulum, mitochondria and Golgi apparatus were observed (Fig. 11b).
Diabetic group: semithin sections showed that most of the endocrine cells of islets of Langerhans had vacuolated cytoplasm and deeply stained nuclei (Fig. 12a). Congested blood capillaries were very commonly seen.
Ultrastructurally, the previous changes were manifested in the B cells by large electron-lucent vacuoles and reduced amount of secretory granules that were surrounded by wide halos (Fig. 12b). Some B-cell nuclei were euchromatic showing less heterochromatin clumps; others were shrunken and deeply stained (Fig. 12b).
Sildenafil-treated diabetic group: semithin sections of islets of Langerhans showed pale-stained nuclei of endocrine cells with prominent nucleoli. The cytoplasm was granular with minimal vacuolation. The blood capillaries were mildly congested (Fig. 13a).
Ultrastructural examination of B cells showed euchromatic nucleus and regions of moderate amount of characteristic dense core granules with clear membrane-bound regions. Small vacuoles of fused granular membranes and lysosomes were observed. Other organelles were more or less similar to the control group (Fig. 13b).
There was significant (<0.05) decrease in the mean count of GCTs, and also in the mean count of EGF-immunolabelled cells in the diabetic group compared with the control group (Tables 1 and 2). The intensity of EGF staining was graded weak in the diabetic group compared with controls (Table 2). In the sildenafil-treated diabetic group, there was significant increase in the mean count of GCTs and EGF-immunopositive cells compared with the diabetic group (Tables 1 and 2). It was possible to observe that the duct cells stained strongly for EGF (Table 2). Histograms 1 and 2 showed that both the mean count of GCTs and EGF-immunopositive cells in the sildenafil-treated diabetic group significantly decreased compared with the control group.
The results of this study demonstrated that glandular EGF expression is profoundly affected in diabetic rats. The combination of increased salivary expression of EGF after sildenafil treatment, strongly positive immunoexpression of EGF receptors in seminiferous tubules and interstitial cells of treated rat testis, together with relative recovery of B cells of islets of Langerhans, strongly suggests the modulatory effect of sildenafil in cases of diabetes. The effect of diabetes on GCTs was first noted by many researchers [15,16]. Granular duct diameters were reduced in alloxan-treated diabetic rats . In mice with streptozotocin-induced diabetes, the size of the tubules was less than half that of the controls . Despite the reduction in size, the overall granular duct cell morphology at the electron microscopic level was unaffected in diabetic animals [18,19]. Such findings were far beyond our results, which demonstrated marked morphological changes observed in semithin sections of GCTs in the form of significant decreased in number, deeply stained nuclei and degranulation. This was evidenced immunohistochemically by weak EGF expression in the cells of the duct system, which was suspected to be due to decreased protein synthesis. Recent findings  described granular cells in the striated ducts of mouse sublingual glands known as granular striated tubules. They had demonstrated that these granular striated tubules, although similar, were not identical to the GCTs of the submandibular gland. As the animals used in our study were rats, the EGF immunoexpression observed most probably belonged to the GCTs.
Diabetes had a profound effect on protein synthesis by mouse granular duct cells [21,22]. This was previously explained by the impairment of EGF synthesis mainly at the pretranslational levels involving transcription and mRNA turnover . As salivary glands' exocrine function was entirely dependent on autonomic nerves, the question had arisen whether diabetes might affect the neural regulation of salivary secretion . The answer was yes; indeed the secretion of polypeptides from the granular ducts is predominantly an adrenoreceptor-mediated ca+-dependant event [24,25]. Therefore, the effect of diabetes on this intercellular signalling pathway indicated that it was possible that the sympathetic nerve function was impaired in diabetic animals leading to degranulation observed in the semithin sections in this study. In contrast, a similar picture was observed by previous researchers  when they treated rats with isoproterenol (a β-adrenergic stimulator) for different intervals leading to a hyperstimulatory state of GCTs, which then caused depletion of cellular stores of mature EGF and granular cell degranulation. Another possibility that was strongly presumed was that diabetes caused changes in membrane fatty acid profiles. This would lead to alteration of cyclic AMP metabolism and generation of intracellular signalling molecules, such as inositol triphosphate and diacyl glycerol, from membrane phospholipids . The effect of diabetes on the granular duct structure was generally believed to be due to impairment of the hypothalamic pituitary axis . Furthermore, hypophysectomy caused regression of GCT cells, and they were restored by the synergistic action of pituitary-dependant hormones .
In this study, EGF receptor was immunoidentified in the testis of the control group at the cells lining the seminiferous tubules and interstitial cells. It was well known that endocrine control of spermatogenesis was shared by many hormones and growth factors. EGF had a potential role in modulating spermatogenesis by acting directly on germ cells or indirectly on the somatic components of the testis . Several arguments about EGF receptor expression had been raised. Previous researchers detected EGF receptor in Sertoli cells of adult and immature rat testes . However, different researchers  reported that EGF did not seem to be expressed in rat testis, although they found positive EGF binding in presumed peritubular cells by Scathard analysis in cultured cells. A recent publication stated that EGF receptor was observed in Leydig cells and peritubular cells of both normal human testis and testis with decreased spermatogenesis . Finally, EGF and EGF receptor are important autocrine and/or paracrine regulators of spermatogenesis . It was well known that EGF elicits its intracellular effects by means of cell surface localized receptor (EGF-receptor) that exhibited tyrosine autophosphorylation activity on ligand occupancy . In this study, immunohistochemical localization of EGF receptor in diabetic rats was weak. A previous study documented that despite the submandibular gland being the richest source of EGF, Leydig cells are the major source in the testis . Recent researchers  noticed fully developed Leydig cells present in streptozotocin-treated diabetic animals, although abnormally distributed. Therefore, we could postulate that there was a sort of EGF receptor downregulation due to EGF produced by Leydig cells compensating for its deficiency in plasma. The observations of a recent research  showed that diabetes suppressed the expression of EGF receptors due to oxidative stress and enhanced the expression of some oncogenes as H-ras and C-fos in an experimental model of chemically induced oral oncogenesis in normal and diabetic rats. We strongly support the theory of oxidative stress because lipid perioxidation products were able to induce various pathogenic intracellular signals leading to cellular dysfunction . Ultimately, this would lead to failure of ligand–receptor interaction.
With regard to B cells of the pancreas, this study confirmed previous findings that alloxan is a diabetogenic agent causing necrosis of B cells leading to symptoms of diabetes . The present ultrastructural observations showed decreased amount of secretory granules, which possessed wide electron-lucent halos. These results were in accordance with a previous study , which also reported small empty membrane-bound vacuoles and extensive B-cell degranulation. The researchers hypothesized that these vacuoles were due to distended vesicular ergastoplasm, distended Golgi or vacuolated mitochondria. Similarly, another research group  described the cytopathology of pancreatic islets in rats with spontaneous diabetes by quantitative morphometric technique, and proofed a diminished volume density of total secretory granules and increase in immature secretory granules and lysosomes of B cells. At a latter study [41,42], impairment of islet cell function in alloxan diabetes was thought to be mediated by reactive oxygen species. Alloxan is selectively accumulated in B cells through uptake by the glucose transporter in the plasma membrane rapidly causing the formation of hydrogen peroxides, superoxides and hydroxyl radicals .
In this study, diabetic rats treated with sildenafil showed a significant increase in number and improvement in the histological structure of GCTs observed in semithin sections at the light microscopic level. The immunopositive cells for EGF showed strong reaction and significant increase in count compared with the diabetic group. These morphologic data supported a previous study , which postulated that sildenafil increased the secretion of total protein and EGF but not amylase from the submandibular gland. The exact mechanism of sildenafil's stimulatory effect is through its antioxidant activities, which might be attributed to its enhancing effect on cellular cyclic nucleotides cyclic AMP and cyclic GMP , thus contributing in protection against oxidative damage in diabetes. Increased lipid perioxidation caused increased reactive oxygen species due to increased glucose concentration . The relationship between sperm lipid perioxidation and cyclic nucleotides in normozoospermic and asthenozoospermic specimens had been reported suggesting the protective role of these enhanced cyclic nucleotides against oxidative stress . Another mechanism for the action of sildenafil could be activation of the nitric oxide (NO) system against oxidative stress through overexpression of NO synthase . Therefore, sildenafil might have compensated the NO deficit caused by loss of neuronal NO synthase occurring in diabetes .
In these results, EGF receptors showed strong positive immunoreactions in diabetic rat testis after sildenafil treatment. This expression we accounted for increased EGF secretion by the submandibular gland, which is directly related to regulation in the amount of receptors on the target cells. Interestingly, B-cell ultrastructure observed in this study was improved after sildenafil treatment, which could be attributed to its antioxidative actions. Recent approaches have used EGF for stimulating B-cell regeneration directly or by EGF-stimulated mesenchymal cells [47,48]. Therefore, it was reasonable for B cells to recover after increased EGF secretion due to sildenafil antioxidative actions. In addition to alloxan properties in being rapidly excreted in urine and it does not concentrate in pancreas for long , so it might not has a constant effect on the B-cells.
From all the previous results, we can definitely conclude that sildenafil had an important role on salivary secretion of EGF and EGF receptor expression in the testis, putting in consideration its antioxidant capabilities in combating diabetic complications, such as male infertility.
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