Vitamin C supplementation during chronic variable stress exposure modulates contractile functions of testicular artery and sperm parameters in male Wistar rats

Humans are exposed to identifiable multiple environmental stressors. However, there is paucity of studies assessing chronic variable stress (CVS) exposure together with antioxidant supplementation on reproductive function. This study investigates impact of vitamin C supplementation, during CVS exposure on contractile functions of testicular artery and sperm parameters. Twenty pubertal male Wistar rats (150–200 g) were grouped into four of five rats each consisting of control (normal saline), vitamin C treated (7 mg/kg bwt), chronic variable stress (CVS) + vitamin C, and CVS only. Animals were treated respectively for 8 weeks. Serum superoxide dismutase (SOD), catalase, malondialdehyde (MDA) activity, testosterone, and cortisol were assayed. Contractile responses of excised testicular artery to phenylephrine, potassium chloride, acetylcholine, and sodium nitroprusside were determined using Ugo Basile data acquisition system. Sperm parameters and histopathology of the testis were also determined. Statistical significance was taken at p < 0.05 MDA activity was significantly increased in group exposed to chronic variable stress. However, groups supplemented with vitamin C during CVS exposure showed no significant increase. Serum SOD and catalase were enhanced in group exposed to CVS with vitamin C supplementation when compared to group exposed to chronic variable stress only. Chronic variable stress exposure increased serum cortisol level and this was attenuated with vitamin C supplementation. Vitamin C supplementation significantly increased serum testosterone level when compared to CVS only exposed group. Contraction responses of testicular arterial strips to phenylephrine and potassium chloride were attenuated in vitamin C supplemented groups when compared to CVS only exposed group. Vitamin C supplementation during CVS exposure also enhanced relaxation of testicular artery strip to sodium nitroprusside and acetylcholine. Sperm count and percentage motility were significantly reduced in CVS only exposed group when compared vitamin C supplemented group. Sperms cells with abnormal morphology were also significantly increased in chronic variable stress only exposed group when compared to control and vitamin C supplemented groups. Chronic variable stress adversely impacts contractility of testicular artery, sperm parameters, cortisol, and testosterone level. Vitamin C supplementation modulated these by suppressing MDA and enhancing antioxidant enzyme activity.


Background
Stress is a state of threatened homeostasis caused by intrinsic or extrinsic adverse forces (stressors) and is counteracted by an intricate repertoire of physiologic and behavioral responses aiming to maintain/reestablish the optimal body equilibrium [1]. Prolonged stress situation with high circulating level of cortisol is reported to upset homeostasis causing harm to organs, tissues, and vulnerability to diseases [2]. Continuous exposure to stress is known to impair testosterone production, sperm, and erectile functions [3]. Inhibition of testosterone biosynthetic enzyme activity by stress-induced increase in serum glucocorticoids is postulated to be responsible for impaired testosterone secretion [4].
Oxidative stress-induced generation of free radicals is associated with diseases like cancer, Alzheimer, and erectile dysfunction [5]. Oxidative stress has been reported to not only cause but also exacerbate erectile tissue impairment through the reaction of superoxide (O 2 −) with nitric oxide (NO), inhibiting relaxation of the corpus cavernosa [6]. Normally, penile cavernous and arterial smooth muscles play key roles in the erectile processes [7]. Hormones and brain nervous impulses are known to guide special nerve cells in the penis to produce nitric oxide [7]. This nitric oxide then relaxes smooth muscles in penile erectile arteries and erectile bodies [7]. Experimental evidences have suggested that arterial insufficiency in penile arteries usually precedes structural and functional changes in erectile tissues leading to organic erectile dysfunction [8,9]. Testicular tissues are predisposed to activity of free radicals and oxidative stress. Asadi et al. [10] identified reasons for this to include high cell division rate, competition by cells for oxygen, and low oxygen pressure due to weakened vessels.
It is known that balance between reactive oxygen species (ROS) and antioxidant capacity is vital in the pathophysiology of disease state [11]. Well-known antioxidants include beta-carotene, lutein, resveratrol, vitamin C, E, lycopene, and phytonutrients [12]. Our bodies produce some antioxidants, although this can be inadequate especially in stressful situation [12]. An increase in oral intake of vitamin C has been proposed to be potentially beneficial in reducing oxidative damage to tissues by chemical reduction of oxidant species [13]. McCabe et al. [14] reported that sustained release of high dose vitamin C reduced anxiety and mitigate increased blood pressure in response to stress. Individuals are exposed to variable stress daily due to identifiable multiple environmental stressors. Apart from adverse implication of chronic stress, its variability has also been postulated to also impact negatively on body's adaptive response [15]. Evidently [16,17], there exists paucity of studies despite prevalence in nature investigating effects of combined stresses on male reproductive functions and the impact of vitamin C supplementation. This study investigated the effect of chronic variable stress exposure with vitamin C supplementation on stress biomarkers, sperm characteristics, and in-vitro contractile responses of testicular artery in adult male Wistar rats.

Animals
Twenty healthy adult male Wistar rats weighing between 150 and 200 g and purchased from College of Health Sciences Ogbomosho, Oyo state, Nigeria were used. Animals were acclimatized for 3 weeks under conventional laboratory conditions (temperature 25-27°C, humidity of 70%, and 12 h light and 12 h dark cycle) and allowed free access to standard diet and water ad libitum. Standard protocol on animal handling and care (according to the declaration of Helshinki) were strictly adhered to. Procedures adopted were also certified by the Lagos State University College of Medicine Animal House Committee.

Stress models procedure
The rats were exposed to variable stress models according to the method of Mueller and Bale [18], with slight modification for predator exposure and noise. Six painless, non-habituating stress models which do not affect feeding were adopted. They include immobility by restraining animal in 50 ml tube for 20 min during light cycle, multiple cage changes at interval of 20 min for 2 h during light/dark cycle, saturated beddings with water overnight causing sleep deprivation, predator exposure by placing a cat in the same cage as the rats with a wire mesh separation, foreign object exposure in cage overnight, and exposure to 100 decibels sound for 4 h during the light cycle.

Experimental design and treatment
Twenty pubertal male Wistar rats (150-200 g) were randomly divided into four groups of five rats each. Groups 1 and 2 were treated with normal saline (vehicle) and vitamin C 7 mg/kg bwt [19], respectively orally. Groups 3 and 4 were exposed to chronic variable stress with group 3 concurrently treated orally with vitamin C (7 mg/kg bwt). Treatments and stress exposure were for 8 weeks in all groups. All animals were weighed weekly using weighing scale (Highland Adam Equipment, UK).

Serum sample collection
Cardiac puncture technique was used (after injecting 30 mg/kg phento barbital) [20] to collect a single, good quality volume of blood into a plain bottle from the animals using a 5 ml syringe with needle. The blood was allowed to stand at room temperature for 15-30 min. Thereafter, it was centrifuged using a cold centrifuge (Model SM112, Uniscope Laboratory Centrifuge, England) at 4000 rpm for 15 min. The resulting supernatant designated serum was carefully aspirated using a Pasteur pipette into a plain bottle and stored at − 4°C

Preparation of the testicular artery strip
The rats were anaesthetized (30 mg/kg phento barbital), before they were sacrificed by cervical dislocation [20]. The testis was surgically removed en bloc with care. The testicular artery was removed from the testis and placed in a petri-dish containing physiological salt solution (PSS). The testicular artery was suspended in a 50 ml chamber of the organ bath. These chambers contained PSS with the following composition (m/mol): NaCl (118.2), CaCl 2 (1.6), KCl (4.7), NaHCO 3 (15.0), KH 2 PO 4 (1.2), MgSO 4 (1.2), and glucose (11.5). The temperature of the organ bath was maintained at 37°C and the solution was bubbled with a 95% O 2 + 5% CO 2 gas mixture (pH 7.35-7.40). Each testicular artery strip was anchored with a stainless-steel hook to an electronic transducer (model 7004; Ugo-Basile Varese, Italy) connected to a data capsule model 17400 for recording isometric contractions [20].

Dose response of testicular artery strip to phenylephrine and potassium chloride
The testicular artery was allowed to stabilize in the physiological solution for 90 min during which it was stimulated three times at 30 min interval with 10 -7 M phenylephrine. Cumulative dose response of the testicular artery to phenylephrine (10 -9 -10 −5 M) and 10-60 mM KCl were thereafter determined. Isometric contractions generated were recorded through the data capsule acquisition system.

Dose response of testicular artery strip to acetylcholine and sodium nitroprusside
The testicular artery was allowed to stabilize in the physiological solution for 90 min during which it was stimulated three times at 30 min interval with 10 −7 M phenylephrine. Cumulative dose response of testicular artery to acetylcholine (10 -9 -10 -5 M) was determined after pre-contraction with 10 -7 M phenylephrine and 60 mmol KCl. The response was ensured to maintain stable level before addition of another dose. Cumulative dose response to sodium nitroprusside (SNP) (10 -9 -10 -5 M) was determined after pre-contracting the testicular artery with 10 -7 M of PE and 60 mmol KCl respectively.

Histological preparation of the testis
Sections obtained after preparations were stained with hematoxylin and eosin stains following clearance in xylene and were subsequently dried at 35-40°C. Photomicrographs were then taken at 200 and 400 magnifications [21].
Determination of serum superoxide dismutase, catalase, and malondialdehyde activity Superoxide activity was assayed using a randox kit (Sigma Chemicals Ltd., USA). Catalase activity was determined according to the method of Aebi [22]. Malondialdehyde (MDA) an index of lipid peroxidation was determined using the method as described by Uchiyama and Mihara [23].

Determination of serum cortisol, testosterone, and sperm parameter analysis
Serum cortisol and testosterone were estimated using enzyme-linked immunosorbent assay (ELISA) method. Cortisol ELISA kit (DRG, USA) and testosterone ELISA kit Monoblind Inc. Lake Forest, CA, USA were used following the manufacturer instruction. Epididymal sperm count, percentage sperm motility, and abnormal sperm morphology were determined as reported by Raji et al. [21].

Statistical analysis
Results were expressed as means ± standard error of the mean (SEM). Statistical analysis was done using graph pad prism version 5.0. Data were analyzed using oneway analysis of variance (ANOVA), Newman keuls test was used as post hoc test, and p values less than 0.05 were considered statistically significant.

Result
Effect of chronic variable stress exposure with vitamin C supplementation on contractile responses of the testicular artery strip to phenylephrine and potassium chloride Contraction responses of testicular arterial strips to phenylephrine (PE) and potassium chloride (KCl) were attenuated in vitamin C supplemented groups when compared to control and variable stress only exposed groups (Figs. 1 and 2).
Effect of chronic variable stress exposure with vitamin C supplementation on relaxation responses of the testicular artery strips to sodium nitroprusside and acetylcholine Vitamin C supplementation enhanced relaxation response of testicular arterial strips to SNP and acetylcholine (ACh) when compared to stress only group (Figs. 3 and 4).
Effect of chronic variable stress exposure with vitamin C supplementation on serum level of malondialdehyde activity, superoxide dismutase, and catalase Malondialdehyde activity was significantly increased (p < 0.05) in group exposed to chronic variable stress only. However, groups supplemented with vitamin C showed no significant increase (Fig. 5). Serum superoxide dismutase activity was enhanced in group exposed to chronic variable stress with vitamin C supplementation when compared to group exposed to chronic variable stress only (Fig. 6). Catalase serum activity was significantly reduced (p < 0.0001) in group exposed to chronic variable stress. These reductions were attenuated with vitamin C supplementation, though not significantly (Fig. 7).

Effect of chronic variable stress with vitamin C supplementation on serum level of cortisol and testosterone
Supplementation with vitamin C attenuated cortisol increase (Fig. 8). Vitamin C supplementation with or without chronic variable stress exposure significantly increased serum testosterone level when compared to control and chronic variable stress only exposed group (Fig. 9).

Effect of chronic variable stress with vitamin C supplementation on average final body weights of animals
At sixth week, average body weight in chronic variable stress only exposed group was reduced significantly (157 g) when compared to control (242 g) and groups supplemented with vitamin C with (246 g) or without (264 g) chronic variable stress exposure ( Table 1).

Effect of chronic variable stress with vitamin C supplementation on sperm parameters
Sperm counts and percentage sperm motility were significantly reduced in chronic variable stress only exposed group. Vitamin C supplemented groups however showed significant increase (p < 0.05) ( Table 2). Percentage of sperm with abnormal morphology was significantly increased in chronic variable stress only exposed group while vitamin C supplemented groups showed significant reduction ( Table 2). Histological screenings showed no visible lesions in testis across all groups (Fig. 10).

Contractile responses of testicular artery
The result from this study showed the in-vitro modulator capability of vitamin C supplementation despite chronic variable stress exposure. As shown in this study, contraction responses of testicular arterial strips to PE and KCl were attenuated in vitamin C supplemented groups when compared to variable stress only exposed group. Vitamin C supplementation also enhanced relaxation of testicular artery strip to SNP and ACh when compared to stress only exposed group. PE and KCl are vasoconstrictors whose mechanism involves calcium release from intracellular stores through inositol triphosphate (IP3) production and calcium influx through L-type calcium channels respectively [24]. Oxidative stress resulting in ROS generation has been implicated in the impairment of complex intracellular endothelial and neuronal NO mechanism. Vitamin C supplementation in this study appear to counteract excessive damage to complex intracellular mechanism associated with stress-induced generation of ROS. The actual intracellular mechanism through which vitamin C treatment was able to suppress this activity is currently unclear.

Oxidative biomarkers activity
In this study, MDA activity was significantly increased in group exposed to chronic variable stress only when compared to control. However, groups supplemented with vitamin C during chronic variable stress exposure showed no significant increase in MDA. Karafakioğlu [25] reported increased levels of MDA in noise only model of stress exposure with lipoic antioxidant supplementation reducing the MDA activity. Similarly, oxidative stress induced by hypoxia in animals was reported to significantly increase MDA activity with selenium supplementation significantly decreasing MDA tissue and plasma activity [26]. It is noteworthy that despite the variability in stress model adopted in this study as compared to these previous studies, vitamin C supplementation was able to suppress MDA activity. Bashandy et al. [27] had reported that increase in ascorbic acid concentration after supplementation is often sufficient to deal with increased free radical production and MDA formation. Though the plasma ascorbate concentration was not determined in this study, ascorbate anion of vitamin C is reported to be the predominant form existing at physiological pH in the blood [28]. The most striking chemical activity of ascorbic acid as reported is its ability to act as a reducing agent, implicated in detoxifying various oxygen radicals invivo [28]. This activity is well exemplified as observed by the suppressed MDA activity of vitamin C supplemented groups in this study. Serum superoxide dismutase and catalase levels were enhanced in groups supplemented with vitamin C when compared to group exposed to chronic variable stress only. The enhanced superoxide dismutase (SOD) and catalase activity was however not significant. It is currently unknown if increase in vitamin C dose might significantly enhanced their activity in CVS exposed group. Kashif et al. [29] had previously reported that pre and post stress treatment of rats with vitamin C helped increase endogenous SOD and catalase level. SOD is an important endogenous first-line antioxidant enzyme against ROS [30]. It acts by catalyzing dismutation of two molecules of superoxide anion to less hazardous hydrogen peroxide and molecular oxygen. Catalase using either iron or manganese co-factor catalyzes the reduction of hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen in order to complete the detoxification process initiated by SOD [30].

Cortisol and testosterone activity
Supplementation with vitamin C attenuated cortisol increase in this study while it increase (p < 0.05) testosterone level. Acute resistant stress [31], acute and chronic sleep-deprived rats [32] had been reported to cause elevated cortisol levels. The increase in cortisol was attributed to hypothalamic-pituitary-adrenocortical (HPA) axis feedback activation [31]. This feedback mechanism is known to be important for stress survival [31].
Although these aforementioned studies were on single stressor model, the slight attenuation in the cortisol levels in group supplemented with vitamin C in this study highlight the modulatory potential of vitamin C during chronic variable stress exposure. Olayaki et al. [32] had earlier reported the ability of vitamin C causing significant decrease in cortisol levels of acute and chronic sleep-deprived stressed rats. Effect of stress on testosterone secretion in literature appears controversial with immobilization stress often reported to cause testosterone suppression [33,34]. The result from this study shows that if stress application is varied and chronic, testosterone secretion is suppressed. This suppression appeared to be effected by elevated cortisols as observed in this study. Orr and Mann [35] had suggested that restraint stress causing increase in plasma glucocorticoids level act via glucocorticoid receptor in testicular interstitial cells to suppress the testicular response to gonadotrophin. Vitamin C supplementation of chronic variable stress exposure in this study however enhanced testosterone secretion; a similar elevation is reported by Damegh [34] though in immobilized stressed rats.

Body weight and sperm parameters
At sixth week, average body weight in chronic variable stress only exposed group was reduced (157 g) when compared to control (242 g) and groups supplemented with vitamin C with (246 g) or without (264 g) chronic variable stress exposure. Jeong et al. [36] also reported lower body weight and reduced food intake in restrained stressed against normal rats. Restraint stress was reported to reduce body weight and food intake by modifying food intake and energy metabolism related genes. The genetic modifications were suggested to be mediated partly by corticosterone. What is pertinent from our present study however is that, supplementation with vitamin C reduced weight loss in chronic variable stress exposed group. In future, specific influence/role of vitamin C on food intake and energy metabolism-related genes is worthy of being investigated. In this study, epididymal sperm count and percentage sperm motility were significantly reduced in chronic variable stress only exposed group while vitamin C supplementation caused a significant reversal. Percentage of sperm with abnormal morphology was also significantly reduced in vitamin C supplemented groups. Reactive oxygen species are involved in the impairment of sperm functions and their elevation plays major role in the etiology of defective sperm functions. Vitamin C supplementation in this study ameliorates defective effect of chronic variable stress on the semen quality. Vitamin C has been reported to protect sperm cells from oxidative stress induced loss of motility and deoxyribonucleic acid (DNA) damage [37,38]. Corresponding data of suppressed MDA activity in vitamin C supplemented group as reported in this study corroborates this finding.

Conclusion
Vitamin C supplementation substantially modulates reproductive risks in sperm characteristics and contractile functions of testicular artery after multiple chronic stressors exposure. Evidence from this study suggests scavenging power of vitamin C against destructive actions of ROS. Future studies should identify role of specific stress gene expression in this modulation.