Asthenozoospermia (AS), an important cause of male infertility, is characterized by reduced sperm motility. Among the aetiologies of AS, inflammation seems to be the main cause. DJ-1, a conserved protein product of the
Asthenozoospermia (AS) is a common cause of human male infertility [
DJ-1, the
DJ-1 and its homologues, sperm protein 22 (SP22) and contraception-associated protein 1 (CAP1), were the first proteins found to be correlated with male infertility [
Ornidazole- (ORN-) treated rats have been a common animal model to study AS in the last several decades [
We enrolled 10 males (aged 21~45 years) diagnosed with infertility in the Department of Andrology, Peking University Third Hospital, Beijing, China, and 10 age-matched control subjects with normal semen parameters. Semen samples of patients with pyospermia or varicocele or with a history of smoking were not obtained for this study. All participants provided informed consent for participation in the study, and the study was approved by the Ethics Institutional Review Board of Peking University Third Hospital, under protocol number 2011SZ016.
Sexually mature male Sprague-Dawley rats weighing 330~370 g at the beginning of the experiment were obtained from Charles River Laboratories Inc. (SCXK (Jing) 2012-0001, Beijing, China). The rats were maintained at a controlled temperature (24 ± 2°C) and housed by group in separate cages (12 h light/dark cycle) with access to food and water ad libitum. All experimental procedures involving the use of animals were approved by the Animal Care and Use Committee of Peking University.
Total sperm protein was extracted as described previously [
Spermatozoa were collected by density gradient centrifugation, using Percoll solution (Sigma, St. Louis, USA) as a medium [
According to the operation manual recommended by Life Technologies Corporation, spermatozoa were resuspended in prewarmed (37°C) BWW containing 200 nM MitoTracker Deep Red FM (MT-DR FM) (Invitrogen, Carlsbad, USA) and incubated at 37°C for 45 min. After staining, the spermatozoa were washed, spotted on glass slides, and air-dried. These preparations were fixed with 4% paraformaldehyde and then permeabilized with ice-cold acetone. After blocking with 10% goat serum, spermatozoa were sequentially incubated with anti-DJ-1 monoclonal antibody (1 : 200, Abcam, Cambridge, UK) or anti-NDUFS3 monoclonal antibody (1 : 200, Abcam, Cambridge, UK) and then goat anti-rat lgG secondary antibody (1 : 200, Alexa Fluor 555 conjugate) or goat anti-mouse lgG secondary antibody (1 : 200, Alexa Fluor 488 conjugate) (Thermo Fisher Scientific Inc., Rockford, USA). Normal rabbit or mouse IgG was used as a negative control. Nuclei were counterstained with Hoechst 33342. All samples were observed by laser scanning confocal microscopy (TCS SP8-Confocal-MP-FLIM, Leica, Mannheim, Germany).
A rat model of AS was generated by intragastric administration of ORN, according to a previously described method with some modifications [
After the last intragastric administration of ORN or the control solution on day 14, the animals were anesthetized with intraperitoneal (i.p.) administration of 0.6% pentobarbital sodium (10 mL/kg). To assess sperm motility, sperm in the cauda epididymides were collected and prepared as described elsewhere [
Sperm counts were evaluated according to an established method [
Rats were deeply anesthetized with 0.6% pentobarbital sodium (10 mL/kg i.p.) and cardiac perfused with normal saline. The testes and sperm were immediately homogenized in ice-chilled lysis RIPA buffer. The following steps were the same as those used for patients’ samples.
Spermatozoa collected from cauda epididymides were resuspended in prewarmed (37°C) phosphate-buffered saline (PBS) containing 200 nM MT-DR FM and incubated at 37°C for 45 min. After staining, the sperm were pelleted again by centrifugation and resuspended in prewarmed PBS. This spermatozoon suspension was spotted on glass slides and air-dried. The nuclei of cells on the slides were counterstained with Hoechst 33342 and mounted with antifade mounting medium. All samples were observed by laser scanning confocal microscopy (TCS SP8-Confocal-MP-FLIM, Leica, Mannheim, Germany).
The analysis of CI enzyme activity in sperm and testes was performed using a CI Enzyme Activity Microplate Assay Kit (Abcam, Cambridge, UK) following the kit protocol. In short, CI was extracted and introduced into a microplate. After incubation at room temperature for 3 h, the microplate was washed, and assay solution was added. Absorbances of samples were tested at 450 nm for 30 min with an interval of 20 s, following the kinetic program in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, USA). Activity was expressed as the change in absorbance per minute per amount of sample loaded into a well. The data are shown as the standardized ratio of the activity in the ORN-treated rats to that in the control animals.
Using the method described above, the testes of rats were removed and homogenized in a glass homogenizer. The protein extract was preincubated with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) and normal goat IgG (M&C Gene Technology Ltd.) to remove nonspecifically adhered proteins. One milliliter of the above tissue lysate (approximately 500
Statistical analyses were performed with GraphPad Prism 6 for Windows. All data were expressed as means ± standard errors of the mean (SEM). One-way analysis of variance (ANOVA) was used to make comparisons between groups.
General parameters, including age of participants, ejaculate volume, sperm density, and percentage of grade A and grade A + B sperm, are listed in Table
General seminal parameters.
Age | pH | Ejaculate volume (mL) | Sperm density (106/mL) | Grade A sperm (%) | Grade A + B sperm (%) | |
---|---|---|---|---|---|---|
Control | 29.90 ± 1.233 | 7.420 ± 0.08406 | 4.140 ± 0.4110 | 57.23 ± 6.306 | 37.75 ± 1.984 | 60.39 ± 2.951 |
AS | 30.30 ± 1.221 | 7.390 ± 0.05859 | 3.640 ± 0.4031 | 59.53 ± 11.85 | 11.95 ± 1.753 |
22.47 ± 2.139 |
Our previous studies confirmed the significant reduction in DJ-1 in the sperm of patients with AS [
Downregulation of NDUFS3 in the sperm of patients with AS. (a) Representative immunoblot showing NDUFS3 expression in human sperm. (b) Quantification of “a.” NDUFS3 expression was normalized to that of GAPDH as a loading control. The results showed that NDUFS3 expression was reduced significantly in the sperm of patients with AS. (c) Immunofluorescence of DJ-1 (green) and NDUFS3 (red) in sperm from patients with and without AS. Sperm nuclei were stained with Hoechst 33342 (blue). Note that NDUFS3 were both downregulated in the sperm of patients with AS and their colocalization in the midpiece of sperm.
In human sperm, the mitochondrial sheath is organized in a helix of approximately 13 gyres surrounding the axoneme in the midpiece. Mitochondria in sperm were stained by MT-DR FM (Figure
Decreased MMP in the sperm of patients with AS. (a) Immunofluorescence of mitochondria in human sperm, shown by MT-DR FM. Mitochondria in the midpiece of sperm were dyed red. Sperm nuclei were stained with Hoechst 33342 (blue). (b) Percentage MT-DR FM-positive sperm. Note that the sperm of patients with AS showed a significant reduction in MMP.
Sperm motility and concentration were analyzed to determine whether the AS rat model was successfully established. A statistically significant decrease in the proportion of sperm with progressive motility was found in the ORN-treated rats (19.8 ± 3.3%) compared to that in the control rats (47.7 ± 3.1%,
Analysis of sperm motility and concentration in control and ORN-treated rats. (a) Progressive motility (grade A + B) of sperm. The percentage of sperm with progressive motility was quantified and is showed in the histogram. Note that ORN induced a statistically significant reduction in the progressive motility of sperm. (b) Sperm concentration. There was no difference in the effect of ORN treatment on sperm concentration between the two groups.
To investigate whether ORN-treated AS in rats was associated with DJ-1 and NDUFS3 expression during spermatogenesis, we examined DJ-1 and NDUFS3 expression levels in AS and control rats. In the Western blotting analysis, DJ-1 was identified as a single band at ~20 kDa (Figure
Decreased expression of DJ-1 and NDUFS3 in ORN-treated rat testes and sperm. (a) Western blotting detection of DJ-1 protein expression in rat testes. (b) Expression of DJ-1 was normalized against that of
Similar to the results of the MMP assay of human samples, the mitochondria in the midpiece of sperm showed different amounts of staining (Figure
Decreased MMP in the sperm of ORN-treated rats. (a) Immunofluorescence of mitochondria in rat sperm, shown by MT-DR FM. Mitochondria in the midpiece of sperm were dyed red, and sperm nuclei were stained with Hoechst 33342 (blue). (b) Histogram showing the percentage of MT-DR FM-positive sperm. Note that the sperm in ORN-treated rats showed a significant reduction in MMP.
Mitochondrial CI is the first catalytic system in the respiratory chain. A specific reduction in mitochondrial CI activity inhibits sperm motility by regulating the NAD+/NADH redox balance. Thus, we tested CI enzyme activity in sperm and testes of AS rats using an assay kit. The CI activity in the sperm of AS rats (37.93 ± 21.51) was significantly reduced compared to that in the control rats (100.0 ± 15.92,
Decreased CI enzyme activity in the testes and sperm of ORN-treated rats. (a) Analysis of the CI activity of sperm in control and ORN-treated rats determined by CI enzyme activity microplate assay kit. CI enzyme activity in sperm was expressed in mOD/min. The histogram shows the ratio of the normalized mOD/min of rats in the ORN group compared to that in the control group. Note that CI activity in AS rat sperm was reduced relative to that in the control subjects. (b) Analysis of CI activity in testes of rats in the control and AS groups. The data indicate that CI activity in the testes of ORN-treated rats decreased significantly compared to that in the control group.
To assess the endogenous association of DJ-1 with the mitochondria complex, proteins from the rat testes were immunoprecipitated with anti-DJ-1 antibody, and the precipitates were analyzed by Western blotting with anti-NDUFS3 and anti-DJ-1 antibodies. Western blotting analysis identified DJ-1 as a single band at ~20 kDa and NDUFS3 as a single band at ~30 kDa (Figure
Direct interactions between DJ-1 and NDUFS3 demonstrated by coimmunoprecipitation. (a) The protein extracts were immunoprecipitated with an anti-DJ-1 antibody or IgG (negative control), and the precipitates were analyzed by Western blotting with anti-DJ-1 and anti-NDUFS3 antibodies. (b) The final coimmunoprecipitation result was obtained by subtracting the amount obtained from that of the negative control. The histogram shows the amount of DJ-1-NDUFS3 complex (presented as the ratio of the amount of normalized coimmunoprecipitated NDUFS3 versus that in the input) in rat testes. Note that DJ-1 and NDUFS3 were shown to interact in rat testes, and the ability of these two proteins to bind was significantly decreased in the testes of ORN-treated rats.
In the present study, we found that the expression of NDUFS3 and DJ-1 was both decreased in the sperm of patients with AS. We established an AS rat model to investigate this association further. CI activity was decreased in the testes and sperm of AS rats. DJ-1 and NDUFS3 expression levels were similarly reduced in the AS rat testes. The expression of DJ-1 was decreased in AS rat sperm. Moreover, a protein interaction between DJ-1 and NDUFS3 was demonstrated in the rat testes for the first time, and this interaction was weakened in AS.
Although the exact etiology of diminished sperm motility is still generally unexplained, ultrastructural defects of the sperm flagellum due to congenital defects and sperm degeneration caused by genital infections, oxidative stress, anti-sperm antibodies, cryopreservation, or metabolic disorders have been implicated [
DJ-1, a protein related to male reproduction and infertility, has pleiotropic functions, ranging from a role as a chaperone with protease activity to that of a transcriptional regulator, redox sensor, and antioxidant scavenger [
Mitochondria generate adenosine triphosphate by oxidative phosphorylation (OXPHOS) via the mitochondrial respiratory chain, which consists of five multisubunit complexes (CI–V) composed of at least 75 nuclear DNA-encoded and 13 mitochondrial DNA- (mtDNA-) encoded proteins [
The similar trends in the expression of NDUFS3 and DJ-1 in the sperm of patients with AS and the testes of AS rats indicated a correlation between these proteins in germ cells. In fact, colocalization of DJ-1 and NDUFS3 has been shown in NIH3T3 and HEK293 cells [
Animal models, historically, have played a critical role in the exploration and characterization of disease pathophysiology, identification of drug targets, and evaluation of novel therapeutic agents and treatments in vivo [
In summary, we have shown a statistically significant reduction in DJ-1 and NDUFS3 expression in patients with AS and in a rat model. Interactions between DJ-1 and NDUFS3 in the testes were demonstrated and suggest that DJ-1 may play a role in maintaining mitochondrial function by means of the association with NDUFS3 during spermatogenesis in the testes. This protective function may be weakened in AS because of a reduction in binding ability as well as a decrease in the amount of DJ-1. This study suggests that downregulation of DJ-1 and NDUFS3 expression likely contributes to mitochondrial dysfunction, which may underlie AS pathogenesis, since current treatments for AS involve in vitro fertilization techniques rather than treatment of male infertility. These findings contribute to a deeper understanding of mitochondrial function in spermatogenesis in AS and may lead to the identification of a new therapeutic target for drug discovery.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Yupeng Wang and Yi Sun contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (no. 81401259).