Effects of vitamin D3 on the level of heat shock protein 70 and oxidative stress in human sperm: a pilot study

Vitamin D3 (Vit.D3) is an antioxidant that can improve sperm motility. However, the specific mechanisms of Vit.D3’s effect on sperm are not yet elucidated. Since heat shock protein 70 (HSP70) and oxidative stress (OS) are effective in sperm motility and it has been shown that vitamin D3 is associated with these factors, thus this pilot study evaluated the effects of Vit.D3 on HSP70 and OS levels in normal and asthenozoospermic semen samples. In this study, the level of HSP70 proteins was evaluated by western blotting and immunocytochemistry in eight normal and nine asthenozoospermic samples. Additionally, malondialdehyde (MDA) assay and nitroblue tetrazolium (NBT) test were used for measuring OS. The results showed that the amount of HSP70 protein was higher in the groups treated with Vit.D3 compared to the control groups. Likewise, in normal and asthenozoospermic samples, OS was increased in treatment groups, but the increase was insignificant in asthenozoospermic samples. According to the data, HSP70 and OS levels were increased in the sperm cells after incubation with Vit.D3; thus, vitamin D3 might improve sperm motility via these factors in vitro.


Background
Infertility is one of the main worldwide concerns in the reproductive health field and it is estimated that 15% of all couples refer to infertility centers annually [1]. Sperm motility disorder is one of the main causes of male infertility [2].
It has been demonstrated that vitamin D3 can enhance sperm motility by regulating intracellular Ca 2+ content [3]. However, the influence of vitamin D3 (Vit.D3) on the other mechanisms that contribute to sperm motility has not been yet fully clarified.
Heat shock protein 70 (HSP70) is a protein that can affect semen quality. This protein can increase cell resistance and preserve cells from harmful effects of stress [4], which by itself can impair sperm motility [5]. Another study showed that a low level of HSP70 expression in sperm led to the decline of antioxidant enzymes' activities and sperm motility. Therefore, HSP70 protein is effective in controlling sperm movements [6].
Another important agent for sperm motility is oxidative stress (OS) [7]. OS is defined as a condition in which oxygen and oxygen-derived free radicals, known as reactive oxygen species (ROS), exceed the physiological level [8]. Controlled concentrations of ROS are required for normal sperm function, but in excessive levels, they overwhelm the body's antioxidant defenses, which could damage DNA, lipids, and proteins and result in impaired motility, concentration, and morphology of sperm. However, antioxidants such as Vit.D3 can react with ROS, assist in preventing OS onset, and preserve the spermatozoa's function [9].
Finally, since HSP70 and OS are effective in sperm motility, this study was conducted to evaluate the effects of Vit.D3 on the levels of these factors in sperm cells of normal and asthenozoospermic men. This was performed in order to characterize some possible mechanisms of Vit.D3 in improving sperm motility, and the results can be used to improve the quality of the reproductive process. Seventeen human semen samples were randomly collected and transferred to the Cellular and Molecular Research Center. After liquidation, samples were loaded on slides for analyzing sperm parameters (count, motility, morphology, and leukocytes percentage) and selecting normal and asthenozoospermic samples according to WHO's 2010 guidelines [10]. It should be noted that samples of men who had underlying disorders such as varicocele and infection, as well as cigarette smokers and those who were taking medications were excluded from this study. Each sample was washed twice with 1 ml fresh sperm wash solution and centrifuged at 300×g for 5 min. Then, 2 ml fresh sperm wash was added to the cell plate which was then kept at a 45°angle in an incubator at 37°C for 45 min in order to have the motile sperms swim up [11].

Vitamin D3 treatment
The supernatant of each sample was divided into two groups: One was considered the control group and the other received 20 μmol of Vit.D3 (vitamin D3 (cholecalciferol) 300,000 IU/ml-Caspian company) as experimental group, and they were incubated for 1 h [11]. After incubation, 5 μl of a well-mixed sample was placed on a glass slide and checked under a microscope at × 400 magnification for sperm motility assessment. According to the WHO's definition [10], motility of spermatozoa is classified into the following groups: progressive motility (PR), spermatozoa moving actively, either linearly or in a large circle, regardless of speed; nonprogressive motility (NP), all other patterns of motility with the absence of progression, e.g. swimming in small circles, the flagellar force hardly displacing the head, or when only a flagellar beat can be observed; and immotility (IM), no movement of spermatozoa whatsoever.

Western blotting
Approximately 1.0 × 10 7 spermatozoa were mixed with lysis buffer (170 mg urea, 56 mg thiourea, 14 mg CHAPS, and 76 mg Tris in 18 ml PBS final volume) and 1X protease inhibitor solution on ice for 1 h and then centrifuged at 16000×g for 20 min. The supernatant was frozen at 80°C. SDS-PAGE was performed on a 12% Bis-Tris gel [12]. Electrophoresis and electrotransfer to PVDF membrane were done for each step at 100 V for 70-100 min. After transfer of proteins, PVDF membranes were blocked with 5% milk powder for 1 h at room temperature while being shaken. In the next step, the membranes were incubated with primary monoclonal anti-HSP70 (sc-373867) (1/500) at 4°C overnight and then with goat anti-mouse IgG-HRP secondary antibody (sc-2005) (1/5000) for 1 h at room temperature with shaking. Finally, the bands were detected by a molecular imaging apparatus. Anti-β actin (sc-47778) was used as internal control [13].

Immunocytochemistry
For detection of HSP70 by immunocytochemistry, several smears were created from the samples and fixed with 4% paraformaldehyde for 10 min. All the steps were carried out at room temperature in a humidified chamber. The slides were incubated for 5-10 min in 0.1-1% hydrogen peroxide and were blocked with 1.5% blocking serum. Then, primary and secondary antibodies (1:200) were applied to each slide for 1 h, respectively. Negative control slides were treated with PBS alone during the primary antibody step; after that, slides were incubated in 1-3 drops of peroxidase substrate. Finally, the slides were checked under a microscope at × 400 magnifications. Then, 100 cells were counted according to the staining intensity measurement method in three classes, including high staining, low staining, and colorless.

Measurement of lipid peroxidation
Lipid peroxidation in sperm cells was evaluated by thiobarbituric acid reactive substances (TBARS) method, which was used to measure malondialdehyde (MDA) level. Initially, sperm samples were lysed by the method of doing rapid freeze to − 80°C and thawing to 35°C for at least three times. Then, the samples were centrifuged at 4000×g for 10 min and the supernatant was used for the MDA assays [14].
Two hundred and fifty microliters of supernatant were added to a solution made from 1 ml of 20% trichloroacetic acid and 1 ml of 0.5% thiobarbituric acid. The mixture was then incubated in a boiling water bath at 95°C for 1 h. After cooling, the samples were centrifuged at 4000×g for 15 min and the MDA content was measured by a spectrophotometer at 532 nm. The MDA intensity of spermatozoa was determined by using various concentrations (5-100 μmol/dl) of tetraethoxypropane as standards and the results were expressed as μmol/dl [15].

NBT test
Nitroblue tetrazolium (NBT) test was employed for assessing the level of intracellular ROS. Briefly, 200 μl of a sperm sample was washed twice with 1 ml PBS at 300×g for 10 min. The washed spermatozoa was resuspended in 100 μL PBS and mixed with an equal volume of 0.1% NBT, and the mixture was shaken (10 mg of nitroblue tetrazolium chloride powder in 10 ml PBS) at 37°C for 45 min. After washing, the intracellular formazan crystals were solubilized in 60 μl of 2 mol/l of KOH and 2 mol/l of DMSO. After 5 min, the resulting color of the supernatant was measured by an ELISA reader at 655 nm [16].

Statistical analysis
Data analysis was performed using SPSS 23.0. All the values were expressed as mean ± standard error. Due to the fact that data distribution was not normal, bootstrap test was used for comparison of mean values according to 95% confidence interval (CI) in 95% level. In order to analyze the results of all the western blot experiments, the protein bands' density was quantified by Image J software.

Semen analysis
For determining normozoospermic and asthenozoospermic semen samples, concentration, morphology, total motility, and progressive motility of the sperms were assessed and the results are listed in Table 1. After incubating the samples with vitamin D3, their progressive motility was checked and the best samples were selected. These results are given in Table 2.

Western blotting analysis of HSP70 protein
Western blotting analysis was performed to evaluate the amount of HSP70 protein in human sperm cells. HSP70 bands were detected in 70 kDa line (Fig. 1). The density of bands was analyzed with image J software program and the results demonstrated that the amount of HSP70 protein in normal and asthenozoospermic samples was higher in the groups treated with Vit.D3 compared to the control groups (CI = 0.0598-1.2735 and CI = 0.01-0.48 for normal and asthenozoospermic samples, respectively) (Fig. 1) (Table 3).

Immunocytochemistry analysis of HSP70 protein
Immunocytochemistry study was carried out to further support the western blotting results. In this method, the sperms of the studied groups were counted in three categories of colored, light, and colorless, based on their color intensity. The results showed that high-staining cells were increased in the group treated with Vit.D3 (CI = 7.596-27.375 in normal samples (Fig. 2) and CI = 10.934-22.141 in asthenozoospermic samples (Fig. 3)). The number of low-staining cells was decreased in these groups (CI = − 19.63 to − 5.75 in the normal group (Fig. 2) and CI = 18.95 to − 6.873 in the asthenozoospermic samples (Fig.  3)). In addition, a statistically insignificant decrease was observed in a number of colorless cells in the treatment group compared to the control group in normal samples (CI = − 8.412to 0.597) (Fig. 2). Conversely, the decrease was significant in the asthenozoospermic samples in the treated group (CI = − 7.0 to − 0.634) (Fig. 3).
The results indicated that Vit.D3 could increase the level of HSP70 protein in sperms of normal and asthenozoospermic samples.

Effect of vitamin D3 on MDA level
The MDA levels of normal and asthenozoospermic samples in the group receiving Vit.D3 as well as the untreated group are shown in Fig. 4. According to the data, the MDA level of the normal samples was significantly higher in the treatment group compared to the control group (CI = 0.00240-0.0336) (Fig. 4). In the asthenozoospermic samples, an insignificant increase was observed in the treated group (CI = − 3.818 to 5.446) (Fig. 4).

Effects of Vitamin D3 on intracellular ROS
ROS generation in normal and asthenozoospermic semen samples was evaluated by the NBT test. Results showed that ROS generation in Vit.D3-treated group was significantly higher in the normal samples (CI = 0.0025-0.048) (Fig. 5); moreover, ROS generation was increased in the treated group in the asthenozoospermic samples, but it was insignificant (CI = − 0.408 to 0.048) (Fig. 5).

Discussion
This is the first study that demonstrates the effects of Vit.D3 on HSP70 protein and OS in human sperm function. Motility is the main parameter of sperm and plays an important role in fertility [1]. It has been shown that Vit.D3 can increase sperm motility by unknown mechanisms [17]. HSP70 is a protein that has been recognized to be effective in sperm motility [18]. In the current study, the results of western blotting and immunocytochemistry analysis showed that Vit.D3 significantly increased the level of HSP70 in normal and asthenozoospermic human sperm samples. Many other studies have been performed in order to understand the relationship between Vit.D3 and HSP70. A number of previous studies have shown that members of the HSP70 family of molecular chaperones are also known to interact with non-proteins, including metabolites of Vit.D3 [19].
Moreover, it has been revealed that Vit.D3 is a nontoxic inducer of HSP70 in the rat kidney. It has been reported that HSP70 expression is affected by Vit.D3 synthesis and VDR concentration [20]. In another study, Wu and colleagues suggested that overexpression of HSP70 enhanced mitochondrial metabolism of 25-OHD3 (Vit.D3) in human kidney cells by enzymes that catalyze the synthesis of 1,25OH2D3 (Vit.D3) [21].  Asthenozoospermic 0.90 ± 0.21 0.60 ± 0.14 0.01-0.48 The data shows the ratio of HSP70 protein optical density to beta-actin in the normal (n = 8) and asthenozoospermic (n = 9) samples  [18]. OS is another agent which could alter the quality of sperm motility [7]. In this study, the results of MDA and intracellular ROS measurements in normal samples showed that OS was significantly higher in Vit.D3treated groups in comparison with the untreated groups. In asthenozoospermic samples, the results showed an increase in the treated groups, albeit it was not significant.
Since this study was the first of its kind, its results were compared with other cells as well. Bhat and Ismail indicated that Vit.D3 (1 nmol and 10 nmol for 24 hours) can improve all the OS-related parameters in muscles by augmentation of superoxide dismutase activity.  Therefore, they suggested that OS is increased in humans with Vit.D3 deficiency [23].
Similarly, it has been reported that pretreatment of keratinocyte cells with calcitriol, the hormonal form of Vit.D3, could protect cells against oxidative and heat stresses. Furthermore, authors stated that the effect of calcitriol was dose-dependent and its physiological concentration was 0.1 nmol [24]. Another study reported that incubation of sperms with low concentrations of H 2 O 2 increases the amount of glutathione (GSH) in the sperm cells. Finally, accumulation of GSH increases the GAPDS activity and consequently improves sperm motility [25].
As already mentioned, Vit.D3 increased OS in this study, which is contradictory to prior studies and there are two possible reasons for this phenomenon: One is that this dose of vitamin D3 does not have an antioxidant effect on sperm cells because its effect is dose dependent [24] and sperm is a sensitive cell with many polyunsaturated fatty acids in the plasma membrane and a small antioxidant mechanism in the cytoplasm [26]. The other possibility is that the increase in OS was small and had a positive effect on sperm cells, as one study has reported that low level of H 2 O 2 can improve sperm motility by increasing GAPDS protein activity [25] and another study has shown that middle and high concentrations of H 2 O 2 disrupt sperm motility due to a decrease in GAPDS activity [27]. Although GAPDS activity was not assessed in this study, the second possibility is more acceptable since sperm motility was in fact increased in the present study.

Conclusion
This study reveals that Vitamin D3 may improve sperm motility by increasing HSP70 protein. Furthermore, this vitamin can probably increase OS to a small degree in sperm cells and perhaps OS acts as a mediator in improving sperm cells motility.