Introduction

Over the last few decades, the physiological effects of exercise on the endocrine system have been confirmed. It has been shown that the hypothalamic-pituitary–gonadal (HPG) axis, depending on the intensity and duration of exercise, responds to physical activity. Although the effects of exercise on the reproductive axis have been extensively studied in females, these effects have not yet been fully examined in male subjects [1].

The type, duration, and intensity of exercise were found to have different clinical outcomes [2]. Several studies have assessed whether exercise can affect male fertility, some showing that the number of sperms and their ability to move decreased in response to exercise [1]. Moreover, it has been suggested that hypogonadotropic hypogonadism is very common among athletes [3].

Several reports have demonstrated that total and free levels of testosterone (T) are increased by physical activity [4]. It should be noted that the effect of exercise duration and intensity, as well as the type of exercise and the subject’s age on T levels, is paradoxical [4, 5]. For example, the total serum levels of T increased in male cyclists (8 h of exercise per week) compared with recreational athletes (who exercised less than 30 min on most days of the week). In contrast, low levels of T were observed in endurance and resistance athletes, soccer players, and amateur cyclists compared with healthy non-exercisers [1]. Furthermore, in a paraclinical study conducted by Bliss et al. (1972), low plasma testicular T levels were also observed in rats after swimming for 1 h [6]. However, running on a treadmill with a progressive 8 weeks protocol did not change basal serum T levels or testicular concentrations of T [7].

Previous studies have indicated that exercise as a stress stimulus [8] can increase the serum levels of cortisol [9], norepinephrine (NEP), and epinephrine (EP). An increase in the serum levels of cortisol is mediated by the hypothalamic–pituitary–adrenal (HPA) axis. Changes in cortisol levels are strongly related to the intensity of exercise.[10] It has been reported that cortisol levels increased during physical activity; however, other studies suggested that intensive exercise can prevent the secretion of this hormone. This inconsistency in results may be due to the differences in sampling time, as cortisol has a circadian rhythm [9].

While moderate and intensive exercise increased cortisol levels in blood, light exercise did not significantly increase blood levels [9]. In addition, an experiment conducted in rats showed that continuous exercise (i.e., more than 21 days) increased corticosterone (CORT) levels. In contrast, 21 and 42 days of exercise significantly decreased serum and hippocampal CORT levels [11].

Physical activity increases the activity of the adrenergic system, which is followed by an increase in plasma levels of catecholamine (NEP and EP) based on the intensity and duration of exercise [9]. NEP is one of the important transmitters that regulate LH secretion. NEP has both inhibitory and stimulatory effects on gonadotropin-releasing hormone (GnRH) neurons. Therefore, intracerebroventricular injection of NEP in the ovariectomized rats treated with exogenous estradiol stimulates GnRH neurons and increases LH secretion, whereas injection into the preoptic area of NEP inhibits LH secretion [12].

Regarding the inconsistencies in the effects of exercise on the HPG axis, in this study, the effect of moderate and intensive short- and long-term exercise on the levels of stress hormones and the HPG axis of adult male rats was investigated.

Materials and methods

Inclusion/exclusion criteria

Inclusion/exclusion criteria for this study were as follows:

Inclusion criteria

  1. 1.

    Healthy rats.

  2. 2.

    Adult male rats aged 2–3 months.

  3. 3.

    Weight of over 200 g and lower than 300 g.

  4. 4.

    No movement problems.

  5. 5.

    Before the experiment, all rats were tested on a treadmill, and those able to run on the treadmill were selected.

  6. 6.

    Male fertile rats (by mating with females).

Exclusion criteria

  1. 1.

    Inability to move on the treadmill.

  2. 2.

    Weight of more than 300 g.

  3. 3.

    Weight of less than 200 g.

  4. 4.

    Age of over 3 months.

Thirty male Wistar rats aged 2–3 months (250 ± 50 g) prepared by the animal house of the Shahrekord University of Medical Sciences, Shahrekord, Iran, were used in this experimental study and kept in individual cages. During the experimental period, the rats were maintained at a temperature of 22 °C under a 12-h light–dark cycle (7 AM to 7 PM) and had free access to water and food.

All animal experimental procedures were performed in accordance with the guidelines for the proper conduct of animal experiments issued by the Ethics Committee of the Shahrekord University of Medical Sciences and performed according to the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health, USA (NIH Publication No. 85–23, revised 1985).

Experimental design

The animals were randomly divided into five groups (n = 6) as follows: (1) no-exercise (with no intervention), (2) RME-1 (regular moderate exercise for 1 month), (3) RME-6 (regular moderate exercise for 6 months), (4) RIE-1 (regular intensive exercise for 1 month), and (5) RIE-6 (regular intensive exercise for 6 months). The body weight of the rats at the baseline and end of the experiment is presented in Table 1.

Table 1 The role of exercise in weight reduction at the beginning and the end of the exercise period
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Training protocol

The experimental rats were exposed to running on a treadmill for 60 min/d and at a speed of 22 and 35 m/min during 5 consecutive days. First, the speed was set at 5 m/min to warm up and gradually increased to 22 m/min (in the moderate exercise group) and to 35 m/min (in the intensive exercise group). During the last 5 min, to cool down the rats, the speed decreased to 5 m/min. The rats in the no-exercise group were placed on the non-moving treadmill for 60 min. It should be noted that all exercised rats were treated before 12 AM [13].

Blood sampling and ELISA assay

Finally, all experimental rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) [14, 15] 24 h after the last day of exercise at 8 to 11 AM. Then, blood sampling was done from orbital sinus and the separated serum was used for the assessment of T (Cusabio-CSB-E05100r; sensitivity range: 0.06 ng/ml), luteinizing (E0179Ra-Bioassay Technology Laboratory; sensitivity range: 0.015mIU/ml), CORT (Cusabio-CSB-E07014r; sensitivity range: 0.1 ng/ml), and NEP (Bioassay Technology Laboratory-E00514Ra; sensitivity range: 2.51 ng/ml) hormones.

Real-time polymerase chain reaction (RT-PCR) technique

The rats were swiftly euthanized 24 h after the behavioral experiments and their hypothalamic arcuate nucleus was immediately removed, placed in liquid nitrogen, and frozen. Using YTzol Pure RNA buffer (Yekta Tajiz, Iran), all RNAs were extracted from the hypothalamic arcuate nucleus samples and their concentration and purity were then detected with the NanoDrop device, and using a reverse transcription kit (BIONEER), RNA underwent the process of reverse transcription. Triple reaction gene-specific primers are used to measure Gnrh mRNA expression with cDNA samples. The sequences of primers and thermal schedule used in this study are presented in Tables 2 and 3. Next, RT-PCR was performed using SYBR Green PCR Master Mix (Takara Bio Inc.). Finally, real-time quantitative PCR analysis was performed using the comparative CT method (2−ΔΔCT method) and the arithmetic formula was used to measure the relative expression of target mRNAs above the reference values [16].

Table 2 The sequence of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and (Gnrh) primers in rats
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Table 3 RT-PCR time and temperature protocols for Gnrh gene expression
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Data analysis

Statistical analysis was performed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Comparison among the various groups was performed using nonparametric tests (Kruskal–Wallis followed by pairwise multiple comparison procedures (Dunn’s test) because of the small sample size (i.e., six rats per group). The results are presented using the median (min, max) and the significance level was set at 0.05 in all analyses.

Results

The changes in CORT levels in response to exercise

Statistical analysis using Kruskal–Wallis followed by Dunn’s test showed significant differences between groups (P = 0.002; Fig. 1). Specifically, there was a significant decrease in serum levels of CORT in the RME-1 group (P = 0.047) and a significant increase in the RIE-1 group (P = 0.040) compared with the no-exercise group. Moreover, a significant increase in the RIE-1 group was observed compared with the RME-6 (P = 0.047) group. Furthermore, the levels of CORT in the RME-6 (P = 0.040), RIE-1 (P < 0.001), and RIE-6 (P = 0.015) groups significantly increased compared with the RME-1group. However, no significant difference was observed in the RME-6 (P = 0.948) and RIE-6 (P = 0.657) groups compared with the no-exercise group (Fig. 1). The CORT levels are presented as median (min, max) in Table 4.

Fig. 1
figure 1

Changes in serum levels of corticosterone between the RME-1 (regular moderate exercise for 1 month), RIE-1 (regular intensive exercise for 1 month), RME-6 (regular moderate exercise for 6 months), and RIE-6 (regular intensive exercise for 6 months) groups (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with the no-exercise group; #P < 0.05, ##P < 0.01, and ###P < 0.001 in comparison with the RME-1 group; $P < 0.05, $$P < 0.01, and $$$P < 0.001 in comparison with the RIE-1 group. Data are expressed as median (min, max) and assessed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test

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Table 4 Descriptive statistics [minimum (min), maximum (max), and median values] of serum concentration of CORT, NEP, LH, T, and hypothalamic mRNA relative level of Gnrh in no-exercise group (with no intervention), RME-1 (regular moderate exercise for 1 month), RME-6 (regular moderate exercise for 6 months), RIE-1 (regular intensive exercise for 1 month), and RIE-6 (regular intensive exercise for 6 months)
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Changes in NEP levels in response to exercise

The comparison of exercise duration effects on the levels of NEP showed that there was a significant difference between groups (P = 0.002; Fig. 2). Specifically, there was a significant increase in the RIE-1 group compared with the no-exercise (P < 0.001) and RME-1 (P = 0.002) groups. Furthermore, this parameter was found to be significantly decreased in the RIE-6 (P = 0.008) and RME-6 (P = 0.024) groups compared with the RIE-1 group.

Fig. 2
figure 2

Changes in serum levels of norepinephrine (NEP) between the RME-1 (regular moderate exercise for 1 month), RIE-1 (regular intensive exercise for 1 month), RME-6 (regular moderate exercise for 6 months), and RIE-6 (regular intensive exercise for 6 months) groups (n = 6). *P < 0.05,**P < 0.01, and ***P < 0.001 in comparison with the no-exercise group; #P < 0.05, ##P < 0.01, and ###P < 0.001 in comparison with the RME-1 group; $P < 0.05, $$P < 0.01, and $$$P < 0.001 in comparison with the RIE-1 group. Data are expressed as median (min, max) and assessed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test

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However, no significant difference was observed in the RME-6 (P = 0.115) and RIE-6 (P = 0.238) groups compared with the no-exercise group (Fig. 2). The NEP levels are presented as median (min, max) in Table 4.

Changes in LH levels in response to exercise

A significant difference was observed in LH serum levels in the experimental groups (P = 0.001; Fig. 3). Dunn’s test indicated a significant reduction in the levels of LH in the RIE-6 group compared with the no-exercise group (P = 0.019). Moreover, LH serum levels significantly decreased in the RIE-6 (P < 0.001) and RIE-1(P = 0.001) groups compared with the RME-1 group. The levels of LH in the RME-6 group were significantly decreased compared with those of the RME-1 group (P = 0.012). However, a significant difference was not observed in the RME-6 (P = 0.512) and RIE-1 (P = 0.184) groups compared with the no-exercise group (Fig. 3). The LH serum levels are presented as median (min, max) in Table 4.

Fig. 3
figure 3

Changes in serum levels of luteinizing hormone (LH) between the RME-1 (regular moderate exercise for 1 month), RIE-1 (regular intensive exercise for 1 month), RME-6 (regular moderate exercise for 6 months), and RIE-6 (regular intensive exercise for 6 months) groups (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with the no-exercise group; #P < 0.05, ##P < 0.01, and ###P < 0.001 in comparison with the RME-1 group. Data are expressed as median (min, max) and assessed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test

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Changes in T levels in response to exercise

The results of the Kruskal–Wallis test showed a statistically significant difference in T serum levels between the experimental groups (P = 0.001; Fig. 4). Pairwise comparisons using Dunn’s test indicated a significant difference between the no-exercise group and RME-1 (P = 0.024) and RIE-6 (P = 0.048) groups. Moreover, the levels of T in the RIE-6 group significantly decreased compared with those of RIE-1 (P = 0.003) and RME-1 (P < 0.001) groups. T serum levels significantly decreased in the RME-6 group in comparison with those of RME-1 group (P = 0.023; Fig. 4). The serum levels of T are presented as median (min, max) in Table 4.

Fig. 4
figure 4

Changes in serum levels of testosterone (T) hormone between the RME-1 (regular moderate exercise for 1 month), RIE-1 (regular intensive exercise for 1 month), RME-6 (regular moderate exercise for 6 months), and RIE-6 (regular intensive exercise for 6 months) groups (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 in comparison with the no-exercise group; #P < 0.05, ##P < 0.01, and ### P < 0.001 in comparison with the RME-1 group; $P < 0.05, $$P < 0.01, and $$$P < 0.001 in comparison with the RIE-1 group. Data are expressed as median (min, max) and assessed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test

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Changes in Gnrh expression in response to exercise

A significant difference was observed between the experimental groups in terms of changes in Gnrh expression levels (P < 0.001; Fig. 5). Pairwise comparisons using Dunn’s test indicated that the level of Gnrh expression significantly increased in the RME-1 group (P = 0.016) and decreased in the RIE-6 (P = 0.029) group compared with the no-exercise group. Furthermore, the level of Gnrh expression in the RIE-6 group significantly decreased compared with the RME-6 (P = 0.003) and RME-1 (P < 0.001; Fig. 5) groups. Also, the expression level of Gnrh significantly decreased in the RIE-1 group compared with the RME-1 group (P = 0.001). The relative values of Gnrh expression are presented as median (min, max) in Table 4.

Fig. 5
figure 5

Changes in the relative expression of Gnrh between the RME-1 (regular moderate exercise for 1 month), RIE-1 (regular intensive exercise for 1 month), RME-6 (regular moderate exercise for 6 months), and RIE-6 (regular intensive exercise for 6 months) groups (n = 6). *P < 0.05,**P < 0.01, and ***P < 0.001 in comparison with the no-exercise group; #P < 0.05, ##P < 0.01, and ###P < 0.001 in comparison with the RME-1 group. Data are expressed as median (min, max) and assessed by the Kruskal–Wallis test followed by Dunn’s multiple comparisons test

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Discussion

In the present study, serum levels of CORT and NEP (as biomarkers of stress responses), LH, T, and hypothalamic mRNA levels of Gnrh were measured in response to various types of exercise. Studying hormonal changes in response to exercise is of great importance since it has been revealed that exercise stimulates changes in the neuroendocrine system and basal concentrations of stress hormones (CORT and NEP) in response to physical activity [17]. Previous studies have indicated that, depending on oxygen consumption, exercise has different effects on the HPA axis and modulates CORT secretion and reproductive ability [18].

The results of the present study showed that RME-1 significantly reduced CORT levels compared with the no-exercise group, which is consistent with previous findings. The beneficial effects of moderate and regular exercise on reducing stress have long been known [19]. For example, regular and moderate-intensity physical activity (such as 8 weeks of walking) reduced urine cortisol levels in depressed women and improved the subjects’ depression [20]. In addition, moderate-intensity physical activity reduced serum cortisol levels leading to stimulating neuroplasticity and serotonin production in rats exposed to stress [21, 22].

Duration and intensity of exercise have different effects on stress hormone levels [17]. Light and moderate exercise reduced the levels of cortisol (in humans) and CORT (in rodents) [19]. On the other hand, other researchers have shown that by increasing the intensity of exercise, the levels of stress hormones are increased [23]. Moreover, there was a significant increase in CORT levels in the RIE-1 group compared with the no-exercise group, which is consistent with our results. However, intense exercise did not increase cortisol levels during the daytime hours [24]. This inconsistency may be due to the sampling time in different studies and cortisol circadian rhythm [9].

The results of the present study showed that CORT levels are affected by the pattern of exercise intensity. Thus, after moderate-intensity exercise, this level decreased, and following an increase in the intensity of exercise, a significant increase was observed. Furthermore, there was no difference in CORT levels between the RME-6 and RIE-6 groups compared with the no-exercise group. This finding confirmed that the HPA axis does not change in response to moderate long-term (i.e., a fixed form of exercise for a long time) and intense exercise [17]. The results of the present investigation showed that an increase in exercise duration, regardless of exercise intensity, may alter HPA axis adaptation, as the serum levels of CORT did not change in the rat exercise groups compared with those of the no-exercise rats.

In addition, in our investigation, serum levels of NEP (as a biomarker of the stress response) were measured in response to exercise. NEP is known to be an indicator of sympathetic system function [25] and exercise activates the sympathetic nervous system. NEP can alter the release of GnRH and LH hormones, which affects reproductive activity.

Based on our results, RME-1 could not increase the level of NEP hormone compared with the no-exercise group. Also, other studies have shown that catecholamine levels did not increase in the low-intensity exercise group compared with the no-exercise group [26], and our results confirmed this finding. However, RIE-1 treatment increased the concentration of NEP compared with the no-exercise group. There is a relationship between exercise intensity and plasma levels of NEP [26]. As the intensity of exercise increases, the levels of NEP are increased. These findings are consistent with the results of the present study.

The sympathetic nervous system is one of the factors that regulate the body’s metabolic and physiological responses and is necessary for the energy required by the muscles involved in exercise [26]. During intense exercise, the muscles need more oxygen and substrate; therefore, the sympathetic nervous system becomes more active. In this study, RIE-1 treatment increased serum levels of NEP probably by stimulating the nervous system. The obtained results showed that in both types of intensity, increased duration of exercise prevented significant changes in NEP levels compared with the no-exercise group. In other words, the serum levels of NEP showed no significant increase in the RME-6 and RIE-6 groups compared with the no-exercise group. This finding demonstrated that the nervous system is adapted to long-term exercise because, by an increase in the duration of exercise, the HPA axis and plasma concentration of NEP remain stable [17]. Long-term exercise increases the half-life of this hormone (in the heart and spleen), while causing a reduction in NEP ability for regeneration (turnover) and reducing the activity of peripheral noradrenergic neurons during intensive exercise, important factors for a constant level of peripheral NEP [27]. Serum levels of LH and T hormones are considered indicators of reproductive ability, which were considered in this research. One of the strengths of this study was examining the effects of different types of exercise on the serum levels of these hormones, given that there are inconsistencies regarding their serum levels following different types of exercise.

Our results indicated that RME-1 increased the serum levels of T compared with the no-exercise group. Moreover, RIE-6 treatment decreased the serum levels of T and LH compared with the no-exercise group. Previous studies have shown that intense exercise reduced reproductive ability [28], LH and T levels [1, 29], and the quality of semen [28]. Therefore, our results confirmed that moderate exercise has beneficial effects on reproductive function [1, 28]. However, some studies have shown a marked reduction in sperm count, motility, morphology, and viability after the swimming exercise in male Sprague–Dawley rats. Furthermore, the serum levels of reproductive hormones, i.e., T and LH, significantly decreased and histological examination of the testes and epididymis revealed defective spermatogenesis [30]. A possible cause of a reduction in testicular weight could be decreased food intake [31, 32].

The RME-1 treatment increased the relative expression of Gnrh, whereas RIE-6 decreased it. It has been suggested that acute high-intensity exercise (> 60% of maximal aerobic capacity) can increase the circulation of cortisol through its inhibitory effect on GnRH and LH hormones, which is followed by a decrease in T secretion from Leydig cells [33]. Although there is no certain consensus, acute and chronic exercise can suppress endocrine functions at the hypothalamic and testicular levels, specifically, the suppression of GnRH and T [34, 35].

Some studies have shown that chronic swimming exercise suppressed the kisspeptin-GnRH signaling pathway, leading to a reduction in the production of male reproductive hormones [30].

Conclusion

Overall, the results of this study indicated that increasing exercise duration returned the NEP and CORT serum levels closer to the no-exercise group levels. Consequently, the present finding demonstrates the ability of the sympathetic nervous system and HPA axis to adapt to long-term exercise. However, exercise intensity and duration have different effects on the reproductive axis. Further, sexual behavioral studies are needed to confirm the various impacts of different exercise protocols on the male reproductive axis.