Glutathione and free sulphydryl content of seminal plasma in healthy medical students during and after exam stress

S. Eskiocak1,4, A.S. Gozen2, S.B. Yapar1, F. Tavas1, A.S. Kilic2 and M. Eskiocak3

Departments of 1 Biochemistry, 2 Urology and 3 Public Health, Trakya University, School of Medicine, Edirne, Turkey

4 To whom correspondence should be addressed. Email: drseskiocak{at}hotmail.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: It has been reported that there is a relationship between stress and infertility. The mechanisms of stress-related semen quality alterations have not been fully elucidated. In the present study, we investigated the effect of examination stress on seminal glutathione and free sulphydryl content and sperm quality. METHODS: Semen samples were collected from 34 healthy volunteers who were students of medical school in the fourth semester just before (stress period) and 3 months after (non-stress period) their final examinations. Their psychological examination stress was measured by the State Trait Anxiety Inventory (STAI) questionnaire. After standard semen analysis, semen samples were centrifuged at 10 000g for 15 min. Glutathione and free sulphydryl concentration of seminal plasma were measured. RESULTS: During the period of examination stress, the glutathione and free sulphydryl content of seminal plasma and the motility index of spermatozoa were significantly lower, whereas the percentage of morphologically abnormal spermatozoa was higher, than during the non-stress period (P<0.001, for all). An association between seminal plasma glutathione and motility index was observed at both periods (P<0.05 and P<0.01, respectively). CONCLUSIONS: This study demonstrated that glutathione and free sulphydryl levels in seminal plasma decreased in subjects undergoing examination stress. Furthermore, poor sperm quality may be due to loss of glutathione and free sulphydryl content of seminal plasma.

Key words: examination stress/free sulphydryl/glutathione/semen quality


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Reactive oxygen species (ROS) such as superoxide anion (O2·), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH) attack cell membrane phospholipids and induce membrane lipid peroxidation in the sperm. Membrane lipid peroxidation and toxicity of generated fatty acid peroxides in the sperm are major causes of decreased sperm function such as the loss of mammalian sperm motility and decreased capacity for sperm–oocyte fusion (Aitken and Clarkson, 1987Go; Aitken et al., 1989Go).

Protection against ROS and prevention of other damage are of critical importance, and can be provided by both enzymatic and non-enzymatic antioxidants. Seminal plasma can protect spermatozoa from the potentially harmful effect of ROS. On the other hand, the very low amount of cytoplasm and insufficient protection by antioxidants render spermatozoa vulnerable to oxidative damage. The relative contribution of seminal plasma enzymes to the antioxidant activity of the reproductive tract fluids may be negligible. Furthermore, much of the antioxidant activity in semen is non-enzymatic, such as taurine, hypotaurine and glutathione (Lewis et al., 1995Go; Geva et al., 1998Go).

An important endogenous antioxidant in humans is the tripeptide glutathione (L-{gamma}-glutamyl-L-cysteinylglycine; GSH), which plays a central role in the defense against oxidative damage and toxins, where it serves as co-factor for glutathione peroxidase and glutathione S-transferases. Furthermore, GSH plays an important role in the protection against damage produced by oxidants, electrophiles and free radicals owing to its ability to react directly with hydrogen peroxide and superoxide anion, hydroxyl and alkoxyl radicals by its free sulphydryl groups. There is evidence that protein glutathionylation may have a role in the control of such processes. For example, phosphorylase, creatine kinase, carbonic anhydrase, ras, glutathione transferase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) and other proteins have been shown to be glutathionylated (Sies, 1999Go). GSH causes increasing activity of GAPDH, which is a central glycolytic enzyme, and this affects cellular energy status (Galli et al., 2002Go). Also, GSH acts to preserve SH groups of protein in the reduced state by means of disulfide interchange (Sies, 1999Go). The maintenance of free protein sulfhydryl groups is important in the proper folding and activity of protein. When present in the extracellular space, GSH is able to react directly with cytotoxic aldehydes produced during lipid peroxidation, such as 4-hydroxynonenal, and thus protect the free sulphydryl groups on the sperm plasma membrane (Seligman et al., 1994Go). Antioxidants such as GSH and N-acetylcysteine can protect against the damaging effect of leucocytes-derived ROS on sperm movement and be of clinical value in assisted conception procedures (Baker et al., 1996Go). GSH and GSH-related enzymes might play a role in sperm quality (Knapen et al., 1999Go).

Stress is one of the most important health and social problems. Psychological stress has long been suspected as having an important impact on infertility. Clinical observations and experimental studies have comprehensively shown that stress suppresses sexual/reproductive function. The stress scores of infertile couples have been found to be higher than those of fertile subjects (Harrison et al., 1986Go; Boivin et al., 1998Go). An inverse relationship between semen quality and psychological stress was found in infertile subjects undergoing IVF (Clarke et al., 1999Go; Pook et al., 1999Go). Fenster et al. (1997)Go claim that semen quality is negatively affected by stress of the death of a family member. As yet, it is not completely understood whether psychological stress is part of the aetiology of infertility as a causative factor or whether it appears as a consequence of the overall infertility problem. Furthermore, there is a lack of knowledge surrounding the biochemical changes that underline the loss in fertilizing potential as a stress condition. Thus, this study was undertaken to determine GSH and free sulphydryl levels of seminal plasma and sperm quality parameters in a recognized human stress model (medical student examination stress).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and samples
Institutional ethics committee approval was obtained in accordance with the principles of The Declaration of Helsinki. The Declaration of Helsinki suggests that informed consent, confidentiality, obligations (prior and post) and a standard of care are required. Written informed consent was obtained from each study subject. This informed consent included the aims, methods, anticipated benefits and potential risks, and the right to withdraw at any time without reprisal.

Semen samples were obtained from 34 healthy [age (mean±SD) 20.18±1.03 years] volunteers, who were medical school students in the fourth semester, just before final examinations (stress period) and then again about 3 months (11.85±1.23 weeks) later, after their vacation (non-stress period). None of them smoked, used caffeine or alcohol, or had a history of renal disease, liver disease, diabetes mellitus or any acute infection; and none was currently taking any medication such as non-steroidal anti-inflammatories, carnitine, anabolic steroids and vitamins, which are known to influence oxidant-antioxidant status.

None of the subjects underwent exercise, dietary therapy or received supplements during the study period. Oligospermic subjects (spermatozoa density<20x106/ml) were excluded from the study in order to eliminate the possible negative effects of unknown additional pathologies and the study was undertaken only in healthy individuals. None of the subjects had any other stress factors within the last 3 months of the stress period. Subjects who were exposed to any other stress factor between the periods were also excluded from the study.

Samples were collected in the clinical facility by masturbation into a sterile glass container, following sexual abstinence for at least 48 h. For internal quality control of semen analysis, spermiograms were carried out by the same two trained observers, as described earlier, according to the World Health Organization (1993)Go guidelines in our urology clinic. After liquefaction of semen (1 h at 37°C), samples were mixed gently and divided into two parts. The first observer always examined the upper part and second observer always examined the bottom part of the sample at each period. Spermiograms included semen volume (ml), sperm density (x106 per ml), sperm motility (%) and abnormal morphologic features (%). A phase-contrast microscope (Olympus) was used for semen analysis. Sperm density was measured in a Neubauer counting chamber. Sperm motility was classified into four categories: rapid progressive motile, borderline progressive motile, non-progressive motile and immotile spermatozoa, and was assayed at exactly 0.5 and 2 h after liquefaction. Total progressive motility was defined as the percentage of rapid progressive motile plus borderline progressive motile spermatozoa. Motility index as a motility quality indicator was derived by the formula (% total progressive motility/100xsperm count) and demonstrated progressive motile sperm density per ml of semen. Morphology was measured by recording the percentage of abnormal forms in the sample. Diff-Quick stain was used for the examination of morphological features. One hundred cells were examined to determine the cells with normal morphology, which was characterized by normal heads, mid-piece and tails.

Biochemical analysis
Semen samples were centrifuged at 10 000g for 15 min at +4°C and then seminal plasma was separated. For determination of GSH concentrations, a precipitating solution was added to seminal plasma to precipitate all proteins in the sample. After centrifugation at 11 000g, for 15 min at +4°C, the clear supernatants were stored at –76°C until analysis. Glutathione content of seminal plasma was assayed by the Beutler (Beutler et al., 1963Go) method. In this system, glutathione is oxidized by 5,5'dithiobis-2-nitrobenzoic acid (DNTB 10 mmol/l; Merck, Darmstadt, Germany), and then 2-nitro-5-thiobenzoic acid is formed, which can be detected spectrophotometrically by a change of absorption at 412 nm. Standard curves were constructed using reduced glutathione (0.25–10 mmol/l; Sigma, St Louis, MO, USA). Three standard solutions containing normal, medium and high GSH concentrations were analysed on the same day and on 10 different days with 20 replicates. The intra- and interassay coefficients of variation were 1.1% and 1.7%, respectively.

Seminal plasma free sulphydryl content was analysed according to the Hu method (Hu, 1994Go). In this assay, the free sulphydryl (-SH) groups in seminal plasma react with DNTB to form a coloured product that can be measured spectrophotometrically. The intra- and interassay coefficients of variation were <1.0% and <1.2%, respectively.

Stress questionnaire
Psychological stress was measured by the State Trait Anxiety Inventory (STAI), which is widely used for assessing state or acute anxiety (Spielberger et al., 1970Go). STAI was completed by all participants after collection of the semen specimens. The STAI asks the subject to describe how he feels ‘right now’ by responding to 20 questions with a 4-point response format from ‘not at all’ (score 1) to ‘extremely’ (score 4) anxious. Total scores range from 20 to 80, with higher scores indicating greater anxiety. This measure has been shown to have high reliability and good construct validity.

Data analysis
All parameters were given as mean±SD. A paired samples t-test was used to estimate differences between the results of stress and non-stress period. The Wilcoxon signed ranks test was also used for analysis when the data were not normally distributed. Correlations between parameters in the stages were also examined by Pearson Correlation test (Dawson-Saunders and Trapp, 1994Go). Kappa analysis was used to estimate interobserver variables for semen parameters (Lilienfeld and Stolley, 1994Go). The criterion for significance was P<0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The results of seminal and biochemical analysis
The mean abstinence times in stress and non-stress periods were 54.50±3.96 h and 53.62±3.72 h, respectively (P>0.05). The kappa values of rapid progressive motility, total progressive motility and abnormal morphology were >0.40 at both stress and non-stress periods. Sperm density and motility index levels were not different between observers during both stress and non-stress periods (P>0.05, all). The values of semen parameters in stress and non-stress periods are shown on Table I. The density of spermatozoa in stress period was significantly lower than those found in non-stress period (P<0.001). The percentage of total progressive and rapid progressive motile spermatozoa and motility index in stress period were significantly decreased as compared with those found in non-stress period (P<0.01, P<0.05 and P<0.001 respectively), whereas the percentage of immotile spermatozoa in stress condition was found to be higher than in non-stress condition at 0.5 h (P<0.01) after liquefaction. The same results were observed at 2 h after liquefaction (data not shown). The percentage of abnormal morphologic sperm was increased in stress period (P<0.001).


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Table I. Semen quality parameters (±SD) during (stress) and after (non-stress) examination (n=34)

 
Stress scores, GSH and free sulphydryl levels of seminal plasma during and after examinations are shown in Table II. Participants reported significantly higher levels of subjective stress during the examination period compared with that after the vacation (P<0.01). The seminal plasma GSH and free sulphydryl levels were decreased during the examination period compared with those found in the non-stress period (P<0.001 for both).


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Table II. Stress scores and seminal plasma GSH and free sulphydryl levels (±SD), during (stress) and after (non-stress) examination (n=34)

 
GSH and free sulfhydryl contents of seminal plasma were reduced by 35.0% and 22.7%, respectively, during the stress period compared with the non-stress period. Similarly, density of spermatozoa and motility index were reduced by 25.5% and 34.0%, respectively, in the stress period. In addition, the percentages of non-progressive motility, immobility and abnormal morphology were increased by 14.4%, 26.9% and 19.4%, respectively.

Correlations between parameters
The correlations between parameters at both periods are shown in Figure 1. In the stress period, the significant decrease in seminal plasma GSH levels were found to be related to poor sperm quality. There was a negative correlation between the level of seminal plasma GSH and the percentage of immotile spermatozoa (r=–0.368; P<0.05) and the percentage sperm with abnormal morphology (r=–0.407; P<0.05); however, positive correlations were found between GSH levels and the percentage of total progressive motile sperm (r=0.382; P<0.05) and motility index (r=0.425; P<0.05). Seminal GSH level was positively associated with seminal free sulphydryl (r=0.603; P<0.001). A positive correlation was observed between the free sulphydryl content of seminal plasma and motility index (r=0.339; P<0.05).



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Figure 1. Correlations between GSH and free sulphydryl group levels and various sperm parameters before and after the examination period.

 
In the non-stress period, there was a positive correlation between seminal GSH level and total progressive motility (r=0.363; P<0.05), rapid progressive motility (r=0.360; P<0.05) and motility index (r=0.456; P<0.01). There was also a negative correlation between seminal GSH level and the percentage of immotile sperm (r=–0.353; P<0.05). A positive correlation was also found between seminal GSH and free sulphydryl (r=0.502; P<0.01) (Figure 1).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Medical students in Turkey must complete an intensive program, especially in their second year. Therefore, stress is very common among the medical students during the course of their training. They are at their most stressed during their final examinations. To investigate a relationship between stress and semen quality, and the effects of stress on seminal GSH and free sulphydryl groups content, we decided to study semen samples from medical students during their final examinations and then again 3 months later, after their vacation. This interval time is necessary, because of the duration of the spermatogenic cycle, which is 74 days in humans (Turek, 2000Go).

Effects of abstinence time on the results of semen analysis were eliminated in this study by the abstinence time of subjects, which was confirmed by the staff when the semen samples were collected. The abstinence time was not different at either of the collection times. Some studies have reported a seasonal variation in density and motility of spermatozoa. It was reported that the highest sperm density was found in spring and lowest in summer (Gyllenborg et al., 1999Go). Chen et al. (2004)Go also reported that sperm concentration in the spring was significantly higher than in winter, fall and summer. They also emphasized that sperm motility and the percentage of sperm with normal morphology was higher in the spring than in any other season. In this study, the first semen samples were obtained at the end of May (stress period) and the second samples were obtained in August (non-stress period). We observed a decrease in sperm concentration and motility during the stress period. Therefore, the observed differences were not due to seasonal effects and abstinence time.

In the present study, in the stress condition density of spermatozoa, motility index, the percentage of total progressive and rapid progressive motile spermatozoa were significantly lower than those found under non-stress conditions (P<0.001, P<0.001, P<0.01 and P<0.05, respectively), whereas the percentage of immotile spermatozoa and abnormal morphology were increased (P<0.01 and P<0.001, respectively). As expected, we observed that during the stress conditions participants had a higher stress score, which is an index of stress level. The results showed that sperm quality parameters were negatively affected under the stress condition. Since the subjects of the present study have not been under any other stress such as illness or a family member's death, our findings suggest that stress alone may have deleterious effects on semen quality.

One study in the literature showed that in the spermatozoa of patients with oligozoospermia, which causes infertility in many cases, the GSH concentration of sperm was significantly lower than in the normospermic subjects (Ochsendorf et al., 1998Go; Bhardwaj et al., 2000Go). Raijmakers et al. (2003)Go reported that median levels of seminal plasma GSH were significantly lower in subfertile males as compared with the fertile males, and found a positive association between seminal GSH level and semen morphology and motility quality. We observed that seminal GSH levels were significantly decreased during the examination period (P<0.001).

We showed that higher GSH levels in seminal plasma were associated with a higher quality of motility and that lower GSH levels were associated with a higher degree of spermatozoa with abnormal morphology and immotility. In both stress and non-stress conditions, GSH content of seminal plasma was negatively correlated with immotility; however, it was positively correlated with total progressive motile spermatozoa and motility index. These data suggest that a lower level of GSH in seminal plasma is associated with poor sperm quality and a higher level of GSH in seminal plasma is associated with good sperm quality. Therefore, our results imply that seminal plasma GSH levels may play a role in the protection against oxidative damage of the spermatozoa and fertility. These emphases are in accordance with previous findings which indicated that exogenous supplementation to semen samples protected sperm against ROS damage and that GSH therapy improved semen quality (Irvine, 1996Go; Lenzi et al., 1998Go).

Lewis et al. (1997)Go found no significant difference in free sulphydryl of seminal plasma between fertile and infertile males. However, Alkan et al. (1997)Go reported that seminal plasma free sulphydryl in infertile patients were significantly lower than those in the control groups. Armstrong et al. (1999)Go proposed that the toxic ROS produced by activated leukocytes would inhibit both sperm movement and ATP production. They reported that the seminal plasma levels of free sulphydryl in the infertile patients with varicocele and subclinical varicocele were significantly lower than those in fertile males. Furthermore, diminished sperm functions were found to be associated with lower levels of free sulphydryl in seminal plasma. Chen et al. (2001)Go observed that patients with varicocele had lower levels of seminal plasma free sulphydryl as compared with those with subclinical varicocele and the control group. In the present study, we observed that in the stress condition seminal plasma free sulphydryl levels were significantly lower than those found in the non-stress condition (P<0.001).

In the present study, there was a positive correlation between GSH and free sulphydryl levels of seminal plasma both in stress and in non-stress conditions (r=0.603, P<0.001 and r=0.502, P<0.01, respectively). In the stress condition, loss of GSH may lead to a decrease in free sulphydryl. We observed that the loss of seminal plasma free sulphydryl was associated with loss of motility index during examination stress (r=0.339, P<0.05). Our results suggest that free sulphydryl plays an important role in sperm quality. In addition, it was found that the loss of sperm motility in asthenozoospermic samples may result from the overoxidation of sperm sulphydryl (Seligman et al., 1994Go). GSH displays maximal staining in the mid-piece and tail region, which are important regions for mobility of spermatozoa (Agrawal and Vanha-Perttula, 1988Go). These findings are supported by the results of the present study.

To summarize, we have demonstrated for the first time that GSH and free sulphydryl in seminal plasma of subjects under stress conditions are significantly lower than those found under non-stress conditions. Furthermore, we observed that GSH content of seminal plasma was linked with free sulphydryl and sperm quality parameters. We conclude that the semen quality was transiently and adversely affected under the stress; however, to demonstrate the relationship between stress and infertility, further investigations are needed.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We would like to thank Mrs Sebahat Molla for her excellent technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 29, 2004; resubmitted on March 19, 2005; accepted on April 7, 2005.





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