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Gene & Protein in Disease MPDZ regulates sperm motility

2.5. Measurements of intracellular Ca concentration to DMEM/high glucose medium supplemented with 5%
2+
in sperm FBS, and the cells were incubated for an additional 24 h at
Sperm were released in HTF medium from dissected 37 °C in a humidified atmosphere containing 5% CO . The
2
cauda epididymides, as described in “Animals and sperm cells were then harvested for Western blotting. The stattic
collection,” and incubated for 45 min at 37°C in a 5% CO inhibitor (2 μM) was added to MPDZ-overexpression
2
cell culture incubator. Then, 5×10 cells were added to GC2 cells for 12 h. After which, the cells were harvested
5
1 mL HTF containing 1 μM Flou-4-AM and incubated for for analysis.
another 30 min at 37°C protected from light. The cells were 2.9. Western blot analysis
then centrifuged at 800 rpm for 5 min at room temperature
and washed three times in Hank’s balanced salt solution The collected sperm, testes, and cell samples were lysed
(HBSS, 1 mL, without Ca , C0218, Beyotime). The sperm and homogenized for protein extraction and western blot
2+
[32]
were then resolved in 400 μL of warmed HTF medium, analysis, as previously described . Briefly, cell proteins
and a 20-μL aliquot of the suspension was evenly pipetted were extracted in radioimmunoprecipitation assay buffer
onto glass slides. After air drying of the glass slides, the cells (RIPA, 50 mM Tris-HCl, pH = 7.4, 1 mM EDTA, 0.9%
were immediately examined using a BX53F fluorescence NaCl, 0.5% sodium deoxycholate, 1% SDS, 1% NP-40),
microscope (Olympus, Japan). Furthermore, a 100-μL treated with ultrasonic for 30 s, and then incubated in an
aliquot of the residual suspension was used to measure ice bath for 30 min. Sperm proteins were extracted in the
[Ca ] using a fluorescence microplate reader (Molecular same way as cell protein extraction after spermatozoa were
2+
i
Devices, USA). fully ground using a pestle in an ice bath. The samples
were then centrifuged (12,000× g at 4°C for 30 min), and
2.6. Measurements of intracellular Ca concentration the supernatant was collected. Protein quantification was
2+
in cell lines performed using the protein assay kit (P0010S, Beyotime)
GC2 cells were digested with trypsin (SH30042, HyClone) according to the manufacturer’s instructions. Proteins
after being transfected with mouse MPDZ plasmids and were denatured with sodium dodecyl sulfate (SDS) loading
vector control for 24 h, washed three times in HBSS, buffer and heated at 95°C for 5 min, and 40 μg was loaded
resolved in HBSS containing 1 μM Flou-4-AM, and into a 10% SDS-polyacrylamide gel electrophoresis gel.
incubated for 30 min at 37°C protected from light. The Electrophoresis was run at 80 V for 30 min and then
cells were then washed three times in HBSS, centrifuged run at 120 V for another 50 min; then the proteins were
at 1000 rpm for 5 min at room temperature, and then transferred to polyvinyl difluoride membranes (Bio-Rad
resuspended in HBSS (0.5 mL). An equal number of cells Transblot; Bio-Rad, USA) at 220 mA for 150 min in ice
was used to measure the level of [Ca ] using a fluorescence bath; then the membrane was blocked with 5% (w/v) dry
2+
i
microplate reader. skim milk in tris-buffered saline containing 0.1% Tween
20 (TBST) for 90 min at room temperature, washed three
2.7. Statistics of the number of litters times with TBST (10 min/time) before incubated with
Eight 8-week-old male mice of each genotype (wild-type the primary antibodies (1:1000 for MPDZ, ab101277,
[W/W), MPDZ-heterozygous [W/K], MPDZ-null mice Abcam), Stat3 (9139T, Cell Signaling Technology), Stat3
[K/K]) were randomly mated with sexually mature wild- Y705 (9145, Cell Signaling Technology), and 1:500 for
type females (male:female = 1:2) and maintained on a CatSper1–4 (bs-23327R, Bioss; orb156278, orb338146,
standard laboratory diet. The number of newborns was and orb338146, Biobyt); 1:20000 for GAPDH (60004-1-
recorded for 6 months. The average number of litters sired Ig, Proteintech) served as a loading control. Protein bands
per male per month was used to evaluate the fertility of were visualized with electrogenerated chemiluminescence
males of each genotype. using the Fusion FX7 system (Vilber, Korea). ImageJ was
used for quantitative grayscale analysis.
2.8. Stat3 interference experiments
2.10. Reverse transcription-polymerase chain reaction
®
Stat3 expression was interfered with by siRNA SignalSilence (RT-PCR) and quantitative RT-PCR (qRT-PCR) analysis
Stat3 siRNA I (6353, Cell Signaling Technology). Stat3
(Y705) phosphorylation was inhibited with a nonpeptidic RNA was isolated from cell lines, mouse sperm, and
selective Stat3 inhibitor (Stattic, 97598S, Cell Signaling testes using the RNAiso Plus reagent (9109, Takara).
Technology). 100 nM siRNA was transfected into MPDZ- The conversion of total RNA to complementary DNA
overexpression GC2 cells for 6 h using Lipofectamine (cDNA) was performed with the reverse transcription
2000 reagent (11668027, Invitrogen) according to the system (RR047A, Takara). RT-PCR was performed using
manufacturer’s instructions. The medium was changed the Takara Taq™ (R001A) following the manufacturer’s

Volume 2 Issue 2 (2023) 3 https://doi.org/10.36922/gpd.397