Multiple PDZ domain protein regulates sperm motility through CatSper channel

Multiple PDZ domain protein (MPDZ) is a component of the crumbs cell polarity complex and is ubiquitously expressed in mammalian tissues. In mouse sperm, MPDZ was identified as a regulator of the sperm acrosome reaction in the past two decades. Here, a relationship between MPDZ and sperm motility was discovered. During breeding, we found that MPDZ-null mice had smaller litter size. The Ca 2+ signal in mouse sperm decreased. Computer-assisted semen analysis revealed that MPDZ-null males had reduced mouse sperm motility. In humans, MPDZ expression is positively associated with sperm motility. Considering the regulatory role of the CatSper channel in sperm motility and fertility, we identified the expression profile of CatSper subunits (CatSper1–4) and found a reduction at both the transcriptional and translational levels in MPDZ-null mouse spermatozoa. However, in vitro , only CatSper1/2 expression was upregulated in MPDZ-overexpressing GC2 cells. Meanwhile, we found an upregulated cell Ca 2+ signal when MPDZ was overexpressed in GC2 cells. Mechanical analysis revealed that MPDZ bound to the signal transducer and activator of transcription 3 (Stat3) and promoted its phosphorylation at tyrosine 705 (Y705) to upregulate CatSper1/2 expression. Inhibition of Stat3 (Y705) phosphorylation or Stat3 expression attenuated the effect of MPDZ on CatSper1/2 expression. These results suggested that MPDZ was responsible for the transcriptional regulation of the CatSper channel, at least in part, to regulate Ca 2+ signal and sperm motility. To the best of our knowledge, this is the first study to highlight the importance of MPDZ acting as a regulator of sperm motility.

The Ca2+ signal plays the most direct regulatory role in sperm motility [4,16,17] . The dysfunction of the CatSper channel specifically and exclusively impairs the Ca2+ signal, leading to male sterility over an extended period [18][19][20][21][22][23] . Although the responses of the CatSper channel to multiple physiological factors in regulating the Ca 2+ signal have been described in detail for many years, the regulation of the CatSper complex at the transcriptional level remains to be fully understood.
The physiological functions of multiple PDZ domain protein (MPDZ) are diverse. MPDZ has been suggested to be responsible for congenital hydrocephalus disease [24,25] , ethanol withdrawal [26] , maintenance of the tight epithelium of the kidney under hypertonic stress [27] , control of angiogenesis [28] , suppression of lung cancer progression [29] , and control of Ca 2+ signaling in the acrosome reaction [30] . Here, we found that MPDZ acted as a regulator of CatSper1/2 expression. MPDZ promoted the phosphorylation of the Stat3 at Y705, a key site for Stat3 entry into the nucleus to transcriptional activity [31] , and upregulated CatSper1/2 expression. Inhibition of Stat3 (Y705) phosphorylation or Stat3 expression attenuated the upregulation effect of MPDZ on CatSper1/2 expression. Furthermore, increased MPDZ expression was likely to be beneficial for human sperm motility and the high level of Ca 2+ in sperm from volunteers, helping us to understand the physiological function of MPDZ in sperm motility to some extent.

Animals and sperm collection
This study was approved by the Ethics Committee for the Animal Research of Army Medical University (Chongqing, China) (AWUMEC2020708). A C57BL/6 mouse colony was maintained in a temperature-and humidity-controlled animal facility, where all mice had free access to water and food. Male mice (8 ± 1 weeks) were sacrificed by performing cervical dislocation. Then testes and epididymides were carefully dissected. The left testes were immediately incubated with Bouin's liquid for 20 h after rinsed in phosphate-buffered saline (PBS) (pH = 7.4); the right testes were frozen with liquid nitrogen as quickly as we could after morphological observation. The cauda epididymides were carefully trimmed to remove adipose and other tissue, rinsed in PBS, and placed in 400 μL of human tubal fluid (HTF) medium (MR-070-D, Millipore). Five cuts were made on each cauda using iris scissors, and the sperm was released into the medium during incubation for 10 min at 37°C. After incubation, the tissue was removed, and the suspension was gently mixed before measuring motility. For each motility measurement, a 10-μL aliquot of sperm suspension was loaded by capillary action using a largebore pipette tip into one chamber (depth of 10 μm) of a Leja slide. At least 5 fields and sperm counts >200 were recorded for each sample. Sperm parameters were measured with computer-assisted semen analysis (CASA) (ELGA, UK). The residual sperm was stored at −80°C for analysis after rinsed in PBS.

Cell lines
The GC2 and HEK-293T (293T) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium Corning) supplemented with 5% fetal bovine serum (FBS) (11011-8611, Zhejiang Tianhang Biotechnology CO., LTD) and maintained in a humidified atmosphere with 5% CO 2 at 37°C.

Measurements of intracellular Ca 2+ concentration in sperm
Sperm were released in HTF medium from dissected cauda epididymides, as described in "Animals and sperm collection, " and incubated for 45 min at 37°C in a 5% CO 2 cell culture incubator. Then, 5×10 5 cells were added to 1 mL HTF containing 1 μM Flou-4-AM and incubated for another 30 min at 37°C protected from light. The cells were then centrifuged at 800 rpm for 5 min at room temperature and washed three times in Hank's balanced salt solution (HBSS, 1 mL, without Ca 2+ , C0218, Beyotime). The sperm were then resolved in 400 μL of warmed HTF medium, and a 20-μL aliquot of the suspension was evenly pipetted onto glass slides. After air drying of the glass slides, the cells were immediately examined using a BX53F fluorescence microscope (Olympus, Japan). Furthermore, a 100-μL aliquot of the residual suspension was used to measure [Ca 2+ ] i using a fluorescence microplate reader (Molecular Devices, USA).

Measurements of intracellular Ca 2+ concentration in cell lines
GC2 cells were digested with trypsin (SH30042, HyClone) after being transfected with mouse MPDZ plasmids and vector control for 24 h, washed three times in HBSS, resolved in HBSS containing 1 μM Flou-4-AM, and incubated for 30 min at 37°C protected from light. The cells were then washed three times in HBSS, centrifuged at 1000 rpm for 5 min at room temperature, and then resuspended in HBSS (0.5 mL). An equal number of cells was used to measure the level of [Ca 2+ ] i using a fluorescence microplate reader.

Statistics of the number of litters
Eight 8-week-old male mice of each genotype (wild-type [W/W), MPDZ-heterozygous [W/K], MPDZ-null mice [K/K]) were randomly mated with sexually mature wildtype females (male:female = 1:2) and maintained on a standard laboratory diet. The number of newborns was recorded for 6 months. The average number of litters sired per male per month was used to evaluate the fertility of males of each genotype.

Stat3 interference experiments
Stat3 expression was interfered with by siRNA SignalSilence ® Stat3 siRNA I (6353, Cell Signaling Technology). Stat3 (Y705) phosphorylation was inhibited with a nonpeptidic selective Stat3 inhibitor (Stattic, 97598S, Cell Signaling Technology). 100 nM siRNA was transfected into MPDZoverexpression GC2 cells for 6 h using Lipofectamine 2000 reagent (11668027, Invitrogen) according to the manufacturer's instructions. The medium was changed to DMEM/high glucose medium supplemented with 5% FBS, and the cells were incubated for an additional 24 h at 37 °C in a humidified atmosphere containing 5% CO 2 . The cells were then harvested for Western blotting. The stattic inhibitor (2 μM) was added to MPDZ-overexpression GC2 cells for 12 h. After which, the cells were harvested for analysis.

Western blot analysis
The collected sperm, testes, and cell samples were lysed and homogenized for protein extraction and western blot analysis, as previously described [32] . Briefly, cell proteins were extracted in radioimmunoprecipitation assay buffer (RIPA, 50 mM Tris-HCl, pH = 7.4, 1 mM EDTA, 0.9% NaCl, 0.5% sodium deoxycholate, 1% SDS, 1% NP-40), treated with ultrasonic for 30 s, and then incubated in an ice bath for 30 min. Sperm proteins were extracted in the same way as cell protein extraction after spermatozoa were fully ground using a pestle in an ice bath. The samples were then centrifuged (12,000× g at 4°C for 30 min), and the supernatant was collected. Protein quantification was performed using the protein assay kit (P0010S, Beyotime) according to the manufacturer's instructions. Proteins were denatured with sodium dodecyl sulfate (SDS) loading buffer and heated at 95°C for 5 min, and 40 μg was loaded into a 10% SDS-polyacrylamide gel electrophoresis gel. Electrophoresis was run at 80 V for 30 min and then run at 120 V for another 50 min; then the proteins were transferred to polyvinyl difluoride membranes (Bio-Rad Transblot; Bio-Rad, USA) at 220 mA for 150 min in ice bath; then the membrane was blocked with 5% (w/v) dry skim milk in tris-buffered saline containing 0.1% Tween 20 (TBST) for 90 min at room temperature, washed three times with TBST (10 min/time) before incubated with the primary antibodies (1:1000 for MPDZ, ab101277, Abcam), Stat3 (9139T, Cell Signaling Technology), Stat3 Y705 (9145, Cell Signaling Technology), and 1:500 for CatSper1-4 (bs-23327R, Bioss; orb156278, orb338146, and orb338146, Biobyt); 1:20000 for GAPDH (60004-1-Ig, Proteintech) served as a loading control. Protein bands were visualized with electrogenerated chemiluminescence using the Fusion FX7 system (Vilber, Korea). ImageJ was used for quantitative grayscale analysis.

Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) analysis
RNA was isolated from cell lines, mouse sperm, and testes using the RNAiso Plus reagent (9109, Takara). The conversion of total RNA to complementary DNA (cDNA) was performed with the reverse transcription system (RR047A, Takara). RT-PCR was performed using the Takara Taq™ (R001A) following the manufacturer's https://doi.org/10.36922/gpd.397 Gene & Protein in Disease MPDZ regulates sperm motility instructions. Primers for the amplification of the MPDZ, Stat3, CatSper1-4, and GAPDH genes are listed in Table S1.
All qRT-PCR experiments were performed using Master mixes qRT-PCR (RR820A, Takara) with a C1000 real-time cycler (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were carried out in triplicate, and the 2 -ΔΔct method was used to determine the expression of interest genes.
Human MPDZ expression in spermatozoa was determined by the Ct value of MPDZ divided by GAPDH (Ct M/G ). The cDNA of each semen sample of volunteers was obtained from Li Yin (Institute of Toxicology, College of Preventive Medicine, Army Medical University).

Statistical analysis
Data were analyzed using SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA) and R language. The correlation between MPDZ expression and sperm motility was assayed using binary logistic regression. The correlations between MPDZ expression and semen parameters, including sperm motility, [Ca 2+ ] i , progressive motility, and fast-forward progressive motility, were assessed using R language. All statistical tests were two-tailed, and a p < 0.05 was considered statistically significant.

Lack of MPDZ led to a decline in sperm motility and male fertility in mice
To explore the role of MPDZ in spermatozoa, MPDZ-null C57BL/6 mice were used in this study [29] . Almost all MPDZnull mice showed no gross abnormalities in appearance or behavior. During breeding, we found the number of litters sired by MPDZ-null (K/K) males were smaller than their wildtype (W/W) or MPDZ-heterozygous (W/K) counterparts ( Figure 1A), suggesting that MPDZ-null genotype resulted in male fertility decline. To explore this phenomenon, we comparatively analyzed the pathological morphology of the ovaries and testes and the testis-weight ratio between the three genotypes of mice. However, no significant differences among the three genotypes were observed ( Figure S1A-C). With the CASA analysis, we found that MPDZ-null mutation led to a reduction in sperm motility (Figure 1) but not in spermatogenesis or total sperm count ( Figure S1B and S1D). Compared to their W/W counterparts, the fractions of sperm with fast and medium motility in MPDZ-null mice were reduced ( Figure 1B and C), accompanied by no obvious change in the fraction of slow motility sperm ( Figure 1D); meanwhile, the fraction of immotile sperm increased ( Figure 1E), indicating that MPDZ was responsible for maintaining sperm motility. Progressive motility (PR) is essential for spermatozoon movement to eggs for fertilization.
We found that the percentage of spermatozoa with PR visibly reduced in MPDZ-null males ( Figure 1F). The average sperm speed (VAP) also decreased in MPDZ-null sperm ( Figure 1G). A decline in the proportion of spermatozoa with non-progressive motility (NP) was detected in MPDZ-null ones though there was no statistical difference ( Figure 1H). Taken together, these results showed that decreased sperm motility was responsible for the reduction in fertility of MPDZ-null male mice.

The beneficial effect of MPDZ expression on human sperm motility
To further explore the roles of MPDZ in sperm motility, we analyzed the association between MPDZ expression and human sperm motility. Sperm samples were divided into two groups: one with sperm motility less than the cut-off value (40%, according to WHO Laboratory Manual for the Examination and Processing of Human Semen, the fourth edition [33] ); the other with sperm motility equal and/or higher than the cut-off value. As shown in Table 1, MPDZ expression positively affected sperm motility with an OR = 1.601 (>1), although without statistical significance. Moreover, a positive association between MPDZ expression and sperm parameters was found in human sperms. MPDZ expression was inclinedly beneficial for high levels of Ca 2+ signal, sperm motility, sperm PR, and sperm rapid PR, but contrary to sperm with low-PR, NP, and immotility ( Figure S2). These results suggested that MPDZ expression was beneficial for sperm motility and a high level of Ca 2+ signal in sperm.

MPDZ regulated Ca 2+ signal in mouse sperm
We found that MPDZ was responsible for the regulation of the cell Ca 2+ signal using the String database ( Figure 2A). MPDZ was functionally relevant with Ca 2+ channel activity, store-operated calcium channel activity, and inositol 1,4,5-trisphosphate. In mouse sperm, the Ca 2+ signal was intensively uncovered in the areas of the head, neck region, and midpiece of the flagellum and weakly in the front of the principal piece of the flagellum ( Figure 2B), which was analogous to the fact that the Ca 2+ signal was determined in the head, neck region, midpiece of human sperm flagellum [16] . The Ca 2+ signal was much lower in MPDZnull spermatozoa than in the W/W or W/K counterparts ( Figure 2C). Meanwhile, in vitro, the Ca 2+ signal increased when MPDZ was overexpressed in GC2 cells ( Figure 2D). These results indicated that MPDZ was involved in the regulation of the Ca 2+ signal in mouse sperm.

MPDZ regulated the expression of CatSper family genes in mice
The CatSper channel is a unique sperm cation channel, which is the most important ion channel for male https://doi.org/10.36922/gpd.397

Gene & Protein in Disease
MPDZ regulates sperm motility fertility [11] . Here, we found that the expression of the CatSper1-4 genes remarkably decreased at both the transcriptional and translational levels in MPDZ-null spermatozoa ( Figure 3A-C). These results indicated that MPDZ was involved in transcriptionally regulating the expression of CatSper1-4 genes. The mouse MPDZ gene was overexpressed in GC2 cells, further supporting the notion. In vitro, the expression of CatSper1/2 increased in MPDZ-overexpressing GC2 cells at both the transcriptional and translational levels, but CatSper3 expression did not obviously change, and CatSper4 did not express in GC2 cells ( Figure 3D-G). To further explore CatSper4 expression, we designed multiple pairs of primers and used mouse testis cDNA as a positive control. We found that CatSper4 did not express in GC2 cells ( Figure S3). Thus, we concluded that MPDZ participated in the regulation of CatSper1/2 expression.

Transcription factor Stat3 played a role in the regulation of CatSper1/2 expression via MPDZ
Given that MPDZ generally acted as a typical scaffolding protein and was localized in the apical membrane [30,34] ,   we screened the transcription factor Stat3, as it could bind to the promoter regions of CatSper1/2 using the JASPAR database (Table S2). To demonstrate the roles of Stat3 in the regulation of CatSper expression via MPDZ, Stat3 expression was determined. We found that the abundance of Stat3 (Y705) phosphorylation, not Stat3 itself, significantly decreased in MPDZ-null mouse testes ( Figure 4A and B). However, the content of Stat3 (Y705) phosphorylation, not Stat3 itself, increased considerably in GC2 and 293T cells when MPDZ was overexpressed ( Figure 4C and D). These results showed that MPDZ promoted Stat3 phosphorylation.
To demonstrate the transcriptional regulatory role of Stat3 in the CatSper genes, we inhibited Stat3 Y705 using a selective inhibitor in MPDZ-overexpressing GC2 cells and found that CatSper1/2 expression decreased significantly ( Figure 4E and F). A similar observation was obtained after the inhibition of Stat3 expression using siRNA in MPDZ-overexpressing GC2 cells ( Figure 4G and H). These results further Gene & Protein in Disease MPDZ regulates sperm motility confirmed that Stat3 was responsible for the process of MPDZ transcriptional regulating the expression of CatSper1/2.

Molecular docking of MPDZ and Stat3
Computational docking methods have been widely used to generate potential protein-protein interaction

Gene & Protein in Disease
MPDZ regulates sperm motility networks [35,36] . We explore the interaction between MPDZ and Stat3 using Cluspro Sever, a popular Web server for the direct docking of two proteins [37,38] . As shown in Figure 5, interactions of MPDZ-Stat3 were observed between the 7 th PDZ domain of MPDZ (PDB: 2IWQ) and mouse Stat3 (PDB: 4ZIA), between the 12 th PDZ domain of MPDZ (PDB: 2IWP) and mouse Stat3 (PDB: 3CWG). According to the substantial number of low energy docked poses cluster in a narrow vicinity of MPDZ-Stat3 complex, we could reasonably assume that there is a well-defined free energy well around the native complex of MPDZ-Stat3.

Discussion
MPDZ is one of the components of the Crumbs complex [39] , which plays an important role in cell migration. Historically, it has been acknowledged that the crumbs complex functioned as a tumor suppressor, as its loss would induce cancer cell migration and invasion by altering cell polarity [40] . Recent work shows that MPDZ inhibits the migration of lung cancer cells by activating the Hippo signal [29] . These results suggest that MPDZ is an important regulator of cell motility. Sperm is a structurally specialized terminal differentiated cell with high motility potential. Here, we show that MPDZ acts as a regulator of sperm motility in mice. MPDZ knockout led to reduced sperm motility, including fast-, medium-, and progressive motility. Meanwhile, MPDZ expression in human sperm tends to show positive correlations with sperm motility, as well as forward progressive motility and fast-forward progressive motility. Moreover, we find that MPDZ is localized in the principal piece of the sperm flagellum ( Figure S4), and we think that it is the structural basis for MPDZ functionally regulating sperm motility. However, this discovery differs from previous studies, which revealed that MPDZ was detected only in the head of mammalian sperm [41][42][43] . This discrepancy in localization may be due to sperm fixation methods or the epitope of proteins used as immunogens and/or the different spatial conformational epitopes [44,45] . Of course, further exploration is required.
Combined with the fact that MPDZ expression is beneficial for sperm motility in human and murine sperm, we suppose that MPDZ could be a candidate for clinical asthenospermia therapy.
The importance of intracellular Ca 2+ signals in regulating sperm motility has been elegantly shown in recent studies. Over the past two decades, direct electrophysiological recordings, together with experimental results involving genetically modified mouse models and human genetics, have confirmed the importance of the principal Ca 2+ -selective plasma membrane ion channel CatSper for the influx of Ca 2+ . CatSper is a unique family of sperm cation channellike proteins expressed exclusively in the testes and function as a polymodal sensor that translates physical and chemical signals in the reproductive tract into a Ca 2+ response [8] . CatSper-mediated Ca 2+ signaling is integrated into various sperm behaviors, including directed chemotactic turns, random turning, and hyperactivation [16] . The activation of the CatSper channel by intracellular alkalinization as well as various physiological ligands and chemicals at the translational level has been elucidated in detail [46] . Here, we find that the MPDZ gene acts as an activator in the CatSper channel by transcriptionally regulating CatSper1/2, thus leading to an increase in the intracellular Ca 2+ signal of GC2 cells. However, the expression of CatSper4 was not detected in GC2 cells. This could be explained by the fact that CatSper4 expression begins in meiotic and post-meiotic sperm cells [47] , while GC2 is a kind of cell derived from spermatocytes before meiosis [48] .
Except for the translational level, transcriptional regulation of the CatSper has attracted more attention recently. The HMG-box family of proteins is required for murine CatSper1 transcription. The murine Catsper1 promoter is responsive to Sox5, Sox9, and the sexdetermining region Y (SRY) [49,50] . The family of proteins Gene & Protein in Disease MPDZ regulates sperm motility with bZIP domains is found to be involved in the activity of the CatSper1 promoter with the CRE elements identified in both human and murine CatSper1 promoters [51] . Here, we find that Stat3, one of the STAT family of transcription factors, is responsible for the transcription of CatSper1/2. These different transcription factor families may play a regulatory role in the temporal expression of CatSper genes to maintain intracellular Ca 2+ signal and sperm motility. However, how MPDZ regulates Stat3 needs further investigation.

Conclusion
We show that MPDZ regulates sperm motility and fertility in mice and human sperm. The molecular mechanism revealed that sperm motility regulated by MPDZ is connected with the transcriptional regulation of the expression of CatSper1/2 subunits by Stat3. MPDZ and Stat3 can create a complex to phosphorylate Stat3 in Y705 ( Figure 5). Our studies enrich the understanding of regulatory mechanisms of sperm motility and the function of MPDZ.

Acknowledgments
We are particularly grateful to the associate chief technician Wuhua Ni (Reproductive Medicine Center, the First Affiliated Hospital of Wenzhou Medical University) for her kind help with analyzing and giving semen parameters of volunteers; Li Yin (Institute of Toxicology, College of Preventive Medicine, Army Medical University) for her kindness in giving us the cDNA of human spermatozoa; Lu Zhang, an assistant professor (Institute of Medical Bioinformatics, School of Basic Medicine, Henan University) and Fengling Wang (Laboratory of Cell Signal Transduction, Henan Provincial Engineering Centre for Tumor Molecular Medicine, School of Basic Medicine, Henan University) for their kind help to analyze the association between MPDZ expression and semen parameters of volunteers using SPSS 20.0 and R language.

Funding
This work was supported by grants from the Natural Science Foundation Project of Chongqing CSTC of China (No. cstc2019jcyj-bshX0036 and cstc2020jcyj-bshX0036) and the National Natural Science Foundation of China (No. 81902711, 31902287 and 81670988).

Conflict of interest
There are no conflicts of interest to declare.

Ethics approval and consent to participate
The ethical approvals for animal and human subjects by the Ethics Committee for the Animal Research of Army Medical University (Chongqing, China, approval ID: AWUMEC2020708) and the Ethics Committee of Wenzhou Medical University (WenZhou, China, approval ID: AF/SC-08/1.0), respectively.

Consent for publication
Not applicable.

Availability of data
Additional data can be obtained from corresponding author following formal request.