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Gene & Protein in Disease MPDZ regulates sperm motility

1. Introduction were sacrificed by performing cervical dislocation.
Then testes and epididymides were carefully dissected.
Reduced sperm motility is not a common disorder but is The left testes were immediately incubated with Bouin’s
the most direct cause of male infertility . Many factors, liquid for 20 h after rinsed in phosphate-buffered saline
[1]
including organic and inorganic constituents, intracellular (PBS) (pH = 7.4); the right testes were frozen with liquid
pH, and alterations in various ion channels, such as Hv1, nitrogen as quickly as we could after morphological
CaV, P2X2, KSper, and CatSper, lead to poor sperm observation. The cauda epididymides were carefully
motility [2-7] . Among these factors, the CatSper channel trimmed to remove adipose and other tissue, rinsed in
directly affects male fertility [8,9] . The CatSper complex PBS, and placed in 400 μL of human tubal fluid (HTF)
comprises a minimum of ten different proteins, which medium (MR-070-D, Millipore). Five cuts were made
consist of four pore-forming subunits (CatSper1–4) and six on each cauda using iris scissors, and the sperm was
[10]
accessory subunits (CatSperβ, γ, δ, ε, ζ, and EFCAB9) . released into the medium during incubation for 10 min
Knockout or loss of any of the CatSper1–4 or CatSperδ at 37°C. After incubation, the tissue was removed, and the
genes can lead to male infertility [11-15] . suspension was gently mixed before measuring motility.
The Ca2+ signal plays the most direct regulatory role For each motility measurement, a 10-μL aliquot of sperm
in sperm motility [4,16,17] . The dysfunction of the CatSper suspension was loaded by capillary action using a large-
channel specifically and exclusively impairs the Ca2+ bore pipette tip into one chamber (depth of 10 μm) of
signal, leading to male sterility over an extended period [18-23] . a Leja slide. At least 5 fields and sperm counts >200
Although the responses of the CatSper channel to multiple were recorded for each sample. Sperm parameters were
physiological factors in regulating the Ca signal have measured with computer-assisted semen analysis (CASA)
2+
been described in detail for many years, the regulation of (ELGA, UK). The residual sperm was stored at −80°C for
the CatSper complex at the transcriptional level remains to analysis after rinsed in PBS.
be fully understood.
2.2. Cell lines
The physiological functions of multiple PDZ
domain protein (MPDZ) are diverse. MPDZ has been The GC2 and HEK-293T (293T) cell lines were purchased
suggested to be responsible for congenital hydrocephalus from the Cell Bank of the Chinese Academy of Sciences
disease [24,25] , ethanol withdrawal , maintenance of the (Shanghai, China). Cells were cultured in DMEM medium
[26]
[27]
tight epithelium of the kidney under hypertonic stress , (10-013-CMR, Corning) supplemented with 5% fetal
control of angiogenesis , suppression of lung cancer bovine serum (FBS) (11011-8611, Zhejiang Tianhang
[28]
progression , and control of Ca signaling in the Biotechnology CO., LTD) and maintained in a humidified
2+
[29]
acrosome reaction . Here, we found that MPDZ acted as atmosphere with 5% CO at 37°C.
[30]
2
a regulator of CatSper1/2 expression. MPDZ promoted the 2.3. Plasmid construction and cell transfection
phosphorylation of the Stat3 at Y705, a key site for Stat3
entry into the nucleus to transcriptional activity , and For overexpression, the plasmids separately contained
[31]
upregulated CatSper1/2 expression. Inhibition of Stat3 the full-length open reading frame of mouse MPDZ
(Y705) phosphorylation or Stat3 expression attenuated the (NM_010820, pDONR223, G134086), human MPDZ
upregulation effect of MPDZ on CatSper1/2 expression. (NM_003829, pDONR223, G166950), mouse Stat3
Furthermore, increased MPDZ expression was likely to be (NM_213659, pDONR223, G136745), and vector control
beneficial for human sperm motility and the high level of (vehicle control) were purchased from Unibio (Hunan,
Ca in sperm from volunteers, helping us to understand China). Plasmids were extracted using the EZgene™
2+

the physiological function of MPDZ in sperm motility to Endofree Express Plasmid Maxiprep kit (PD1522,
®
some extent. Biomiga). Cells were transfected using a Neon kit
®
(MPK10096) on a Neon Transfection System following
2. Materials and methods the manufacturer’s instructions, and the parameters were
set as follows: Pulse voltage, 1150 V; pulse width, 30 ms;
2.1. Animals and sperm collection pulses, 2 times.
This study was approved by the Ethics Committee 2+
for the Animal Research of Army Medical University 2.4. Intracellular Ca concentration measurements
(Chongqing, China) (AWUMEC2020708). A C57BL/6 Fluorescence measurement of the intracellular Ca
2+
2+
mouse colony was maintained in a temperature- and concentration ([Ca ] ) was performed using Fluo-4-AM
i
humidity-controlled animal facility, where all mice had (λex = 496 nm, λem = 516 nm, S1060, Beyotime) following
free access to water and food. Male mice (8 ± 1 weeks) the manufacturer’s instructions.
Volume 2 Issue 2 (2023) 2 https://doi.org/10.36922/gpd.397