HAN Mingming, DING Fuhong, MENG Zhen, and LEI Jilin*

1)Fisheries College,Ocean University of China,Qingdao266003,P. R. China

2)Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Science,Qingdao266071,P. R. China

Oxidants and Anti-Oxidants in Turbot Seminal Plasma and Their Effects on Sperm Quality

HAN Mingming1),2), DING Fuhong2), MENG Zhen2), and LEI Jilin1),*

1)Fisheries College,Ocean University of China,Qingdao266003,P. R. China

2)Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Science,Qingdao266071,P. R. China

In this research, the concentration and activity of oxidants and anti-oxidants in turbot semen, and their effects on sperm quality were studied. The results showed that superoxide dismutase (SOD), catalase, glutathione reductase (GR), uric acid, vitamin E (VE) and vitamin C (VC) were more abundant in seminal plasma than in spermatozoa. The variation for each of them was specific. In seminal plasma, the activity of SOD and GR increased from November 15, November 30 to December 15, and then decreased on December 30. The concentrations of both VCand uric acid decreased during the first 3 sampling times and increased on December 30. The oxidants in seminal plasma accumulated to the highest on December 30. Lactic acid (LA) and ATP levels decreased to the lowest on December 30. The correlation analysis showed that GR had the significant positive relevance to sperm motility and VSL/VCL, while ·OH had negative relevance to them.

anti-oxidant; seminal plasma; sperm quality

1 Introduction

Increased reactive oxygen species (ROS) concentrations can impair membrane fluidity, decrease sperm motility and damage the genetic material in spermatozoa by base degradation, point mutation, DNA fragmentation and cross-linking of proteins for example, which can result in decreased semen quality and sperm motility, even the loss of their ability of fertilizing the oocytes (Agarwalet al., 2005; Bucaket al., 2010; Sharmaet al., 2004).

The antioxidants in the seminal plasma may provide the protection to the sperm (Franzet al., 2010; Sikka, 2004). Several effective antioxidant systems in the seminal plasma, including SOD, catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) have been studied thoroughly (Bucaket al., 2007). In teleost fish species, early studies have demonstrated that the seminal plasma have anti-oxidative function (Ciereszkoet al., 1999; Ciereszko and Dabrowski, 2000). In rainbow trout, SOD, ascorbic acid (VC) and uric acid have been considered as important semen antioxidants with higher concentration in seminal plasma than in blood plasma (Ciereszko and Dabrowski, 1995; Metwally and Fouad, 2009; Ciereszkoet al., 1999). In brown trout semen, Franzet al. (2010) found that uric acid and catalase showed a positive effect on sperm viability and supposed that uric acid maybe the main antioxidant since it could increase the quantity of spermatozoa with intact membranes, improve sperm motility and swimming velocity, and decrease LPO (lipid peroxidation) in sperm plasma membrane. Besides these, the glutathione and methionine antioxidant systems also exist in the seminal plasma (Franzet al., 2010). Franz and Mansour also found the superoxide dismutase and uric acid had the highest activity and concentration among the antioxidants and oxidant defensive enzymes in semen ofLota lota,Perca fluviatilis,Alburnusalburnus, andSalmo trutta. They concluded that the antioxidant systems in semen of the four species were quite uniform with only minor inter-specific differences (Franz and Mansour, 2010).

As an important commercial fish species, turbot is drawing more and more attentions. Our previous study analyzed the turbot sperm with Computer Aided Semen Analysis (CASA) and found that the sperm quality varied during the four sampling times, Nov. 15, Nov. 30, Dec. 15 and Dec. 30, 2011 (Hanet al., 2013). In this experiment, the activities and concentrations of oxidants /antioxidants in turbot seminal plasma and their effects on sperm quality were studied. The sperm quality variation was tested according to the authors’ previous study (Hanet al., 2013). The quality parameters included semen volume, motility rate, straight line velocity (VSL), curvilinear velocity (VCL), VSL/VCL, seminal plasma protein (spp) content, sperm protein (sp) content, lactic acid (LA) and ATP levels. The anti-oxidants included SOD, catalase, uric acid, vitamin E, vitamin C, and GR. And the oxidants include H2O2, hydroxyl radicals (OH·) and the oxidizedglutathione (GSSG).

2 Materials and Methods

2.1 Collection of Materials

The culture of male turbot and the semen collection followed authors’ previous study (Hanet al., 2013). The semen was collected in sterile EP tube, and stored in an ice box. Semen was centrifuged at 3000×g at 4℃ for 10 min to separate seminal fluid and spermatozoa. The supernatant of seminal fluid was collected in the sterile EP tube. The spermatozoa were homogenized in 1 mol L-1Tris-HCL, pH7.8 at a rate of 1：10 for 2-3 min. After being centrifuged at 3000×g for 10 min, the supernatant was collected into sterile EP tubes, and both the supernatant of seminal plasma and spermatozoa were separated and stored at -80℃.

2.2 Sperm Quality Analysis

The sperm quality was analyzed with Computer Aided Semen Analysis (CASA) and sperm concentration was calculated following our previous study (Hanet al., 2013). The concentration of seminal plasma protein and sperm protein were analyzed with the Coomassie Brilliant Blue G-250 staining kit. Bovine serum albumin was used as the standard according to the manufacturer’s instructions.

The LA and ATP levels in the supernatant of sperm homogenate were analyzed. The LA concentration was analyzed with Vassault method (Vassault, 1983). The analysis of ATP concentration was instructed with the phosphomolybdic acid colorimetric method from the supporter. The absorbance was read with an ultravioletvisible spectrophotometer at 636 nm.

2.3 Analysis of Antioxidants and Oxidants in Sperm and Seminal Plasma

The supernatant of spermatozoa and seminal plasma stored at -80℃ freezer was incubated at 4℃ before analysis. All parameters were determined strictly following the instructions of the kit manufacturer (Nanjing Jiancheng Industry, China).

According to the direction of manufacturer, in the presence of phenanthroline, both vitamin E and vitamin C can reduce Fe3+to Fe2+, and Fe2+will react with the phenanthroline to form pink compounds, after colorimetry, the content of VEand VCcan be figured up with the standard curve of formula.

With the H+provider, NADPH, glutathione reductase can reduce oxidized glutathione (GSSG) to reduce glutathione (GSH). Through detecting the drop-out value of absorption at 340 nm of NADPH and calculating, we obtained the activity of the glutathione reductase (GR). Catalase activity was detected with a spectrophotometer (Beers and Sizer, 1952).

Superoxide dismutase (SOD) can catalyze the superoxide anion (O2-.) into oxygen and hydrogen peroxide (H2O2). O2-.can oxidize hydroxylamine to nitrite. At the end, an amaranth reaction occurs when color agent exists. In this study, control group and experimental group were set. For the experimental group contained SOD, and less nitrite was produced. Finally, the absorbance of it was lower than the control group, and the activity of SOD was obtained at 550nmwith the formula supplied by the manufacturer.

Hydrogen peroxide (H2O2) can react with molybdic acid and also with Fe2+to produce a kind of complex and Hydroxyl radicals (·OH), respectively, and the content of H2O2and ·OH can be determined at 405nmand 550nm.

Through DTNB reaction, the absorbance of GSH and GSSG can be obtained at 405nm, and their concentration can be figured up according to the standard curve.

2.4 Statistics

Data are presented as mean ± standard deviation (n=5). Analysis of variance (ANOVA) was used to find if the concentration of antioxidants, oxidants and sperm quality changed significantly. The correlation analysis was also performed to analyze if there is a close relevance among antioxidants, oxidants and sperm quality with the ‘Analyze-Correlate-Bivariate’ function of SPSS 16.0 (P＜ 0.05 andR＞ 0.7 were considered to be significant).

3 Results

3.1 The Activity and Concentration of Antioxidants and Oxidants in Semen

The activity of anti-oxidants (SOD, catalase, GR, vitamin C and uric acid) and oxidants (H2O2, ·OH and GSSG) are shown in Table 1.

The variation for each antioxidant was specific. In seminal plasma, the activity of SOD increased significantly during the first 3 sampling times and reached the highest on Dec. 15 of 123.01 U mL-1and then decreased to 36.22 U mL-1on Dec. 30. The difference was significant during the experiment (P＜ 0.05). GR and vitamin C both got their highest points on Dec. 15, and then decreased to a lower point. Catalase activity varied with no significance during the experiment.

In sperm, except for ·OH, all of the antioxidants and oxidants changed with no significance in activity or concentration during the experiment.

3.2 Changes in Sperm Quality

Seminal plasma protein (SPP) content, sperm protein (SP) content, lactic acid (LA) and ATP levels are shown in Table 2.

Sperm protein concentration remained nearly constant during the four stages, while seminal plasma protein concentration (mg mL-1) varied significantly during the 4 stages and decreased from 34.56 to 13.09 (P＜ 0.05). LA concentration varied between 10.02 and 12.57 mmol L-1with no significant difference (P＞ 0.05). ATP concentration increased from 24.5 to 40.97 μmol g-1protein in the first 3 stages, and decreased to 3.21 μmol g-1protein on Dec. 30, with the significance only appearing betweenDec. 15 and Dec. 30 (P＜ 0.05).

3.3 The Relevance (Rvalue) Between (Anti-)oxidants and Sperm Quality

As shown in Table 3, among the 5 antioxidants, GR had the significant positive relevance to motility (R= 0.903,P= 0.000) and VSL/VCL (R= 0.855,P= 0.000). Both of hydroxyl radical (Rr= -0.811,P= 0.001) and hydrogen peroxide (Rp= -0.836,P= 0.001) had an negative relevance to VSL/VCL and the sperm motility rate.Notes：?These data were from author’s previous study (Hanet al., 2013). Data are mean ± standard deviation (n=5). The results marked with different letters were significantly different (P＜ 0.05).

Table 1 Activity and concentration of oxidants and anti-oxidants in turbot semen at four sampling times

Table 2 The sperm quality of turbot at four different sampling times

Table 3 Correlations between anti-oxidants and sperm quality (n=12)

4 Discussion

In this experiment, we determined several kinds of anti-oxidants and oxidants in turbot semen from Nov. 15 to Dec. 30 with the intervals of two weeks. Based on the authors’ previous study and with correlation analysis, we also explored the relationship between antioxidants and sperm quality.

Our results showed that the anti-oxidants and oxidants were more abundant in seminal plasma than in sperm, which was necessary for protecting the seminal plasma (Sikka, 2004). In seminal plasma, to anti-oxidative enzymes, SOD had the higher concentration than GR, and CAT, which was similar with the results found in brown trout (Franzet al., 2010). To the anti-oxidative metabo-lites, the concentration of vitamin C was higher than uric acid and vitamin E. Vitamin E was only found in the first two sampling times in seminal plasma, which needed more works to confirm. Though uric acid was considered the most important antioxidant in brown trout semen (Franzet al., 2010), this conclusion was inconsistent with our results. The correlation analysis showed that uric acid had no significant relevance to sperm motility, VSL/VCL, LA and ATP.

Generally, we found that the anti-oxidants reached the lowest concentration and activity on Dec. 15, while the oxidants had the highest content on Dec. 15. The sperm quality also showed the same variation in our previous study (Hanet al., 2013). We hypothesized that during the oxidants scavenging process, though part of the oxidants were scavenged by the anti-oxidants at the beginning, as experiment proceeded, more of the oxidants were produced or accumulated because more damages were caused to anti-oxidants and this prevented the oxidantsscavenging process (Agarwalet al., 2005; Bucaket al., 2010; Sharmaet al., 2004).

SOD and uric acid had the highest concentrations among the anti-oxidants in semen of the four species,L. lota,P. fluviatilis,A. alburnus, andS. trutta(Franz and Mansour, 2010), while through correlation analysis, we found GR had a highest positive relevance to sperm motility and VSL/VCL than others. According to glutathione peroxidase/reductase system, glutathione peroxidase (Se-GSH-Px) could act with glutathione (GSH) as the electron donor to remove reactive oxygen species (ROS) from various peroxides including H2O2, which is responsible to the lipid per-oxidation (Raghuveeret al., 2010). But with the lowest content of GR, the efficiency of it among the antioxidants need more work to find out.

Both ·OH and H2O2had a negative relevance to sperm motility and VSL/VCL according to our correlation analysis. With the highest concentration of oxidants and lowest content of LA and ATP on Dec. 30, we inferred that as experiment proceeded, more oxidants accumulated And then this caused more damage to the intact membrane and broke up the energy production chain. Finally, the production of ATP and LD was hindered or stopped. With less energy provided, leading to the sperm had the poor motility.

In a conclusion, the turbot seminal plasma contained more anti-oxidants and oxidants than sperm, the activity and concentration of the anti-oxidants varied as sperm quality changed. Among the several kinds of antioxidants, GR had a much higher relevance to motility and VSL/ VCL than others.

Acknowledgements

We thank the Tong Yong Aquaculture Industry for providing the male brood stock. We also thank those workers who collected the semen for us, and thank Dr. Guodong WANG for his support to our experiment. This study was supported by the Special Funds of Modern Agriculture Industry System Construction (CARS-50), the National Natural Science Foundation of China (3110188) and the Natural Science Foundation of Shandong Province (ZR2011CQ004).

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(Edited by Qiu Yantao)

(Received November 13, 2013; revised January 6, 2014; accepted March 31, 2015)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015

* Corresponding author. Tel： 0086-532-85821347 E-mail： leijl35@126.com