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. 2012;7(3):e33471.
doi: 10.1371/journal.pone.0033471. Epub 2012 Mar 28.

Effects of hepatitis B virus S protein exposure on sperm membrane integrity and functions

XiangJin Kang  1 , QingDong Xie, XiaoLing Zhou, FangZheng Li, JiHua Huang, DongLing Liu, TianHua Huang
Affiliations

Effects of hepatitis B virus S protein exposure on sperm membrane integrity and functions

XiangJin Kang et al. PLoS One. 2012.

Abstract

Background: Hepatitis B is a public health problem worldwide. Viral infection can affect a man's fertility, but only scant information about the influence of hepatitis B virus (HBV) infection on sperm quality is available. The purpose of this study was to investigate the effect of hepatitis B virus S protein (HBs) on human sperm membrane integrity and functions.

Methods/principal findings: Reactive oxygen species (ROS), lipid peroxidation (LP), total antioxidant capacity (TAC) and phosphatidylserine (PS) externalization were determined. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays and flow cytometric analyses were performed. (1) After 3 h incubation with 25 µg/ml of HBs, the average rates of ROS positive cells, annexin V-positive/propidium iodide (PI)-negative cells, Caspases-3,-8,-9 positive cells and TUNEL-positive cells were significantly increased in the test groups as compared to those in the control groups, while TAC level was decreased when compared with the control. The level of malondialdehyde (MDA) in the sperm cells exposed to 50 µg/ml of HBs for 3 h was significantly higher than that in the control (P<0.05-0.01). (2) HBs increased the MDA levels and the numbers of ROS positive cells, annexin V-positive/PI-negative cells, caspases-3, -8, -9 positive cells and TUNEL-positive cells in a dose-dependent manner. (3) HBs monoclonal antibody (MAb) and N-Acetylcysteine (NAC) reduced the number of ROS-positive sperm cells. (4) HBs decreased the TAC levels in sperm cells in a dose-dependent manner.

Conclusion: HBs exposure could lead to ROS generation, lipid peroxidation, TAC reduction, PS externalization, activation of caspases, and DNA fragmentation, resulting in increased apoptosis of sperm cells and loss of sperm membrane integrity and causing sperm dysfunctions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HBs-induced ROS generation in sperm cells.
A: Comparison between the control, 25 µg/ml of HBs exposure, 25 µg/ml HBs MAb (pretreated)+25 µg/ml HBs exposure, and 1 mg/ml NAC (pretreated)+25 µg/ml HBs exposure(** P<0.01). B: HBs exposure caused dose-dependent increase in ROS production in sperm cells. The data are representatives of five independent experimental replications (five individuals).
Figure 2
Figure 2. Dose-dependent effects of HBs exposure on lipid peroxidation of sperm plasma membranes.
The sperm cells were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h. The MDA levels rose with increasing concentrations of HBs. The data are representatives of five independent experimental replications (five individuals).
Figure 3
Figure 3. Dose-dependent effect of HBs exposure on the total antioxidant capacity in sperm cells.
Sperm cells were exposed to HBs (0, 25, 50, 100 µg/ml) for 3 h. The TAC levels declined with increasing concentrations of HBs. The data are representatives of five independent experimental replications (five individuals).
Figure 4
Figure 4. Effects of HBs on PS externalization in sperm cells.
Sperm cells were unexposed (A) or exposed to 25 µg/ml HBs for 3 hours (B). Cells were stained with FITC annexin V and PI, and analyzed by flow cytometry. Unexposed cells were primarily FITC annexin V and PI negative, indicating that they were viable and not undergoing apoptosis (A). After a 3 hour exposure (B), there were primarily two populations of cells: Cells that were viable and not undergoing apoptosis (FITC annexin V and PI negative) and cells undergoing apoptosis (FITC Annexin V positive and PI negative). A minor population of cells were observed to be FITC annexin V and PI positive, indicating that they were in end stage apoptosis or already dead. The effects of HBs exposure on PS externalization in sperm cells showed dose dependence. (C).
Figure 5
Figure 5. Effects of HBs exposure on caspases-3, -8, -9 activations in sperm cells.
The flow cytometry frequency histograms showed the caspases-3, -8 and -9 activation distributions for the sperm cells unexposed (5A, 5C, 5E) and exposed to 25 µg/ml HBs for 3 h (5B, 5D, 5F), respectively. Cells were labeled with 1 µl of FITC-DEVD-FMK, FITC-IETD-FMK and FITC-LEHD-FMK for 1 h, respectively followed by washing and analysis by flow cytometry. All the histograms of the number of events (Y-axis) versus the fluorescence intensity (X-axis) showed two peaks. In the individual experiment, there were a markedly increase of the caspases-3, -8 and -9-positive cells (53.01%, 45.34% and 59.31%) in the exposed cells when compared with the unexposed cells (40.59%, 28.10% and 36.29%), indicating that HBs exposure was able to induce caspases-3, -8 and -9 activation in sperm cells.
Figure 6
Figure 6. Effects of HBs exposure on caspases-3, -8, -9 activations in sperm cells.
The relationships between the dose of HBs exposure and caspases-3, -8, -9 activations in sperm cells were investigated. The effects of HBs exposure on caspases-3, -8, -9 activations in sperm cells showed a manner of dose dependence. (6A, 6B, 6C).
Figure 7
Figure 7. The effects of HBs exposure on oxidative DNA damage in sperm cells.
HBs-induced oxidative DNA damage in sperm cells in the control group (A) and the test group (B) was assessed by flow-cytometric analysis using TUNEL assay. The rates of TUNEL-positive sperm cells with nuclear DNA strand breaks read from the M2 marker, but the cells without fragmented DNA (TUNEL-negative) from the M1 marker. The x-axis: FL1 channel - the intensity of fluorescence in the green spectrum, the y-axis: depicts the frequency in terms of the number of cells; the fluorescence intensity scale is expressed as “channel number” (0–104). The effects of HBs exposure on oxidative DNA damage in sperm cells exhibited dose-dependence (C).
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