Patients
Thirty cases of male sterility, diagnosed with asthenozoospermia at the General Hospital of Beijing Military Region (Beijing, China) from June 2015 to January 2016, were selected. After 3–5 days of abstinence, 3 ml semen samples obtained by masturbation were analyzed by a sperm quality analyzer, BD-8000G (Xuzhou City Beidou Technology & Trading Co., Ltd., Xuzhou, China). With reference to World Health Organization (WHO) standards on the diagnostic criteria of asthenozoospermia, the percentage of forward moving sperm (grade A + B) was <50% or grade A sperm movement was <25%. The age range of patients was 21–33 years, with an average age of 26.4±5.5 years. At the same time, 30 healthy controls with normal semen samples, according to physical exam were chosen, aged 20–34 years, with an average age of 26.2±5.3 years. The age difference between the two groups was not statistically significant (P>0.05).
The present study was approved by the Ethics Committee of the General Hospital of Beijing Military Region and we received informed consent from all participating patients.
Percoll density gradient centrifugation for sperm collection
One and a half milliliters of 90% and 1.5 ml of 45% Percoll solutions were added to 15 ml centrifuge tubes (90% below, 45% above). Subsequently, 3 ml of semen was added. After centrifugation (200 × g for 20 min), the supernatant was removed and sperm sediment at the bottom was washed with IVF-20 culture medium twice. The sperm precipitate was collected for later use.
Detection of JAK, STAT and PSMβ3 mRNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using RNA Pure, a high purity, total RNA, rapid extraction TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA), and cDNA was synthesized after assessing the purity and concentration of RNA. The primer concentrations were 10 pmol/µl. Primers were designed using Primer Premier 5.0 and were produced by Sangon Biotech Co. Ltd. (Shanghai, China). Primer sequences used were: JAK forward, 5′-TGCTGTCCAGACAAGAATGC-3′ and reverse, 5′-TTCTGCAACCGTCTCTTCCT-3′; STAT forward, 5′-TAACGAGGAGCTGGTGGAGT-3′ and reverse, 5′-GCTTGCGTGTCAGAAAAGTT-3′; PSMβ3 forward, 5′-GAAATCGCAGCCATAGTATC-3′ and reverse, 5′-CTGATGACGGTGAACTTCTG-3′; and β-actin forward, 5′-ATGTTTGAGACCTTCAACAC-3′ and reverse, 5′-GGCCATCTCTTGCTCGAAGTC-3′. According to the Applied Biosystems StepOne system, the reaction mixture contained 10 µl of 2X Smart Green PCR mix, 0.2 µl of each 10 pmol/µl primer, 1 µl of cDNA template, and 8.6 µl of ddH2O for a total volume of 20 µl. The thermal profile was 40 cycles of pre-denaturation at 95°C for 10 min, denaturation at 94°C for 15 sec, 60°C for 1 min and extension at 72°C for 30 sec. The 2−∆∆Cq relative quantitative method was used to show the relative expression levels of each target gene.
Measurement of p-JAK, p-STAT and PSMβ3 by western blot analysis
Each well of the plate was treated by 200 µl lysis buffer and maintained in an ice bath for 1 h. Sample solutions were then centrifuged for 15 min at 4°C. Protein concentration of the centrifuged supernatant was determined by Coomassie Brilliant Blue staining, and preserved at −80°C. Total protein (50 µg) from each sample was separated by electrophoresis. The samples were then transferred to PVDF membranes (90 V, 1.5 h at low temperature). Rabbit anti-human p-JAK (dilution: 1:500; cat. no.: ab138005), rabbit monoclonal p-STAT (dilution: 1:500; cat no.: ab32143) and mouse monoclonal PSMβ3 (dilution: 1:500; cat no.: ab128094), purchased from Abcam (Cambridge, MA, USA), were added after blocking and incubated at 4°C overnight, following four washes with phosphate-buffered saline (PBS). Horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution: 1/2000; Abcam; cat no.: ab6721) was added to membranes and incubated at 4°C overnight. After three washes with PBS, the ECL enhanced chemiluminescence reagent kit was used for signal development. After thorough scanning of negative film, a semi-quantitative analysis was carried out on the bands by Shanghai Tianneng gel imaging processing system software. Data were normalized to levels of β-actin.
JAK inhibitor intervention
Cases without asthenozoospermia were randomly divided into the non-treated control group (n=15) and JAK inhibitor-treated group (n=15). The levels of PSMβ3 mRNA and protein were measured after application of the JAK inhibitor, AG-490. Immunofluorescent staining of PSMβ3 was observed by a laser confocal microscope. The cells were stained as follows: 25-min incubation with 0.25% H2O2, 15 min with 0.3% Triton X-100, three washes with PBS for 5 min, and 30-min incubation with 10% normal goat serum. Rabbit anti-human PSMβ3 monoclonal antibody (1:200) was then added and allowed to incubate at 4°C overnight. The samples were washed three times for 5 min, and biotin-labeled goat anti-rabbit IgG (1:100) was added and left to incubate at room temperature for 30 min. Three 5 min washes with PBS were performed again, and streptavidin-biotin complex-fluorescein isothiocyanate (SABC-FITC)-labeled secondary antibody (1:100) was added, and incubated at room temperature for 30 min. Next, we performed three washes with PBS for 5 min. The samples were stained with DAPI for 5 min, washed in PBS, and mounted using antifade mounting solution. Staining was observed under a confocal microscope (Nikon, Tokyo, Japan). For the blank control group, PBS was used instead of PSMβ3 antibody as described above.
Statistical analysis
Data were analyzed using SPSS 19.0 statistical software (Chicago, IL, USA), and quantitative data are presented as mean ± standard deviation. Comparisons between groups were analyzed by one-way ANOVA, and pairwise comparisons were analyzed using LSD or Bonferroni test. Differences were considered to indicate a statistically significant difference when P<0.05.