V-ATPase Deficiency Aggravates Hypoxia-induced Spermatogenesis Reduction by Promoting Spermatocyte Apoptosis via the JNK/c-Jun Pathway in Mice


 Spermatocyte apoptosis is the primary cause of poor outcome after hypoxia-triggered spermatogenesis reduction (HSR). The vacuolar H+-ATPase (V-ATPase) has been found to be involved in the regulation of hypoxia-induced GC-2 cells apoptosis. However, the mechanism of V-ATPase regulating spermatocyte apoptosis after HSR hasnot been well elucidated. In this study, HSRmodel was established by hypoxia exposure in vivo in V-ATPase-knockout (V-ATPase-/-) and wild-type (WT) mice to investigate theeffectof V-ATPase deficiency on spermatocyte apoptosis. GC-2, amouse pachytene spermatocyte-derived cell line, was introduced in vitro experiments. The sperm count and spermatogenic apoptosis were recorded after 60 d of hypoxia exposure in HSR model. The apoptosis of GC-2 cells was detected by flow cytometry and TUNEL staining. The expression of JNK/c-Jun was evaluated by RNA-seq or western blot. The expression of DR5 and caspase-8 was evaluated by RT-qPCR and western blot. The expression of V-ATPase was determined by western blot in the presence and absence ofLenti-transcription factor EB (TFEB).C-Jun interference was used for evaluating the role of JNK in regulating the apoptosis of GC-2 cells byTUNEL and flow cytometry. The in vivo results suggested that hypoxia induced spermatogenesis reduction and downregulation of V-ATPase. Moreover, V-ATPase deficiency resulted in moresevere spermatogenesis reduction after hypoxia exposure. The spermatogenesis reduction was associated with exacerbation of spermatocyte apoptosis. Hypoxia down-regulated the transcription of V-ATPase through inhibiting TFEB and its nuclear translocation. The mRNA and protein expressions of V-ATPaseincreased after TFEB overexpression in GC-2 cells. Moreover, V-ATPase deficiency enhanced JNK/c-Jun activation and related DR-apoptotic pathwayin GC-2 cells.However,inhibition of c-Jun attenuated V-ATPase deficiency-induced GC-2 cells apoptosis in vitro and HSR in vivo. In conclusion, JNK/c-Jun was involved in the enhancement of V-ATPase-mediated HSR in V-ATPase -/- mice. V-ATPase deficiency aggravates spermatocyte apoptosis, which may account forthe poor spermatogenesis outcomes of V-ATPase-/- mice. The discoveredfunction of V-ATPase modulating spermatocyte apoptosis indicates its potential therapeutic effect against HSR.


Introduction
Male infertility is a multifactorial pathological problemthat affectsapproximately 7% of the male population [1]. The pathogenesis of these male diseasesis usually attributed tothe below two factors:genetic factors [2,3] and environmentalfactors. Among the environmental factors, the published literaturesindicated that hypoxia induced-apoptosis of spermatocyte is the central link of male reproductive dysfunction [4,5]. However, the underlying mechanism of hypoxia induced-apoptosis of spermatocyte remains largely unclear.
Transcription factor EB (TFEB), a member of the bHLH transcriptionfactor TFEB-MITF family, is a master regulator of lysosome biogenesis and autophagy-related gene transcription [6,7]. Starvationregulates the biogenesis of V-ATPase subunits by decreasing mTORC1activity, which subsequently facilitates the entry of the TFEB into the nucleus andthen induces a series of V-ATPases transcription [8].

Mice
All protocols were approved by the Third Military Medical University Institutional Animal Care and Use Committee and conducted following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised in 1978). The V-ATPase KO mice were purchased from Nanfang Model Organisms Center, Shanghai, China. Hypoxic mice were raised in a hypobaric chamber, where the atmospheric pressure was reduced to simulate an altitude of 6000 m. The partial pressure of nitrogen falls as total pressure declines on ascent, but the nitrogen percentage in the atmosphere does not change. Hypoxic mice were returned to normobaric conditions for 40 min every day. Control mice were raised at an altitude of 300 m out of the hypobaric chamber. All the animals had free access to standard pellet food and water, and they were maintained under controlled lighting conditions (12 h light:12 h darkness), ambient temperature maintained at 22-24 o C, and relative humidity of 40-60%.
Epididymal sperm count Mice were sacri ced by an overdose of anesthesia.The epididymis was removed and weighed when dry. Then, they were divided into two parts, head-body and cauda. The method described by Vega et al. was used for sperm count of epididymis cauda [31]. In brief, Caudal regions from the epididymis were isolated and minced. Sperm cells were allowed to disperse out of the cauda. Sperm suspensions were prepared by mincing cauda in 2 mLof phosphate-buffered physiological saline (PBS, pH = 7.2). The suspension was pipetted and ltered through 80 µm nylon mesh to remove tissue fragments. An aliquot (0.05 mL) from the sperm suspension (1 mL) was diluted with 1:50 PBS (PBS, pH 7.2) and mixed thoroughly. An aliquot of the mixture (50 µL) was analyzed for sperm concentration and progressive motility using a Sperm Class Analyzer (Microptic S.L., Barcelona, Spain). The sperm concentration per µLwas multiplied by 50 to obtain total sperm count.

Haematoxylin and eosin (HE) staining
Animals were euthanizedby decapitation. The left testis was excised, xed in 10% formalin, dehydrated, and embedded in para n. Sections were cut at 5 µm thickness and were stained with HEfor lightmicroscopic observations. TUNEL assay Para n-embedded sections were cut into thin slices (4 µm) and depara nized. Nuclei were processed according to the manufacturer's instructions of a TUNEL assay kit (Beyotime). Brie y, the sections were permeabilized with 0.1% Triton X-100,followed with uorescein isothiocyanate (FITC)-labeled TUNEL staining for 1 h at 37 o C. Nuclei were then counterstained with DAPI. The total number of TUNEL-positive nuclei and spermatogenic cells were counted under laser scanning confocal microscope (IX81, Olympus, Tokyo, Japan) and quanti ed as the number of green spots in each photograph (×200) with the assistance of ImageProPlus software.10 photographs were counted.

RNA-seq
After being treated with hypoxia (1% oxygen concentration) or normoxia(21% oxygen concentration) control for 48 h, GC-2 cells were collected and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA,USA). RNA-seq analysis was performed by Personalbio Genomic Technology Co Ltd (Shanghai, China) [32]. Brie y, total mRNA was extracted by a Dynabeads mRNA DIRECT Kit (Ambion).
Thereafter, the cDNA library was constructed using NEBNext Ultra TM Directional RNA Library Prep Kit for Illumina (New England BioLabs Inc.). After fragmentation and conjugation with sequencing adapters, the cDNA libraries were sequenced on Illumina Hiseq 2500 platform using PE100 strategy. The raw data from RNA-seq were ltered and mapped to a reference genome (Mus_musculus.GRCm38.90. chr) by using FASTX. The FPKM (fragments per kilobase of exon model per million reads mapped) was calculated from the ltered and mapped clean raw reads. Furthermore, we employed the tximport package with default parameters to remove abnormal low-/high-abundance transcripts from the transcriptomic pro les. Eventually, the DEGs were obtained from DESeq2 analysis and used for further bioinformatics study.The raw data are accessible toBioProject PRJNA608077 with SRA accession number SRP256125 (experiments SRR11534018-SRR11534023). The raw data set and expression values used for surprisal analysis are made public to enable critical or extended analyses.

Bioinformatics analysis
The sequencing results were processed and the clean reads from the RNA-seq libraries were mapped to the mouse reference genome (Mus_musculus.GRCm38.90. chr) with HISAT2 v2.1.0. Differential expression analysis of six samples was performed using DESeq2. We identi ed differentially expressed genes (DEGs), based on the criteria that P < 0.05 and |log2 (fold change) | > 1.5. The P-values were adjusted using the Benjamini and Hochberg method. The volcano plot of DEGs and cluster diagrams were prepared by R packages "ggplots" and "ggplot2," respectively. KEGG pathway was based on the Kyoto Encyclopedia of Genes and Genomes database, and it was performed to explore the pathways related to these DEGs [33]. A P value of less than 0.05 was considered as statistically signi cant. The enrichment ratio of these pathways was ranked based on the normalized enrich scores (NES). The coe cient r in each gene set was calculated by the Pearson correlation.

Western blotting
After hypoxia treatment, total protein was extracted using a Western & IP cell lysis kit (Beyotime)containing1mM PMSF. Nuclear and cytoplasmic proteins were separated using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) according to the manufacturer's instructions.Whole cell extracts were then separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidenedi oride (PVDF) membrane (Beyotime). Afterblocking, the RNA isolation and real-time polymerase chain reaction Total RNA was extracted from treated cells using 1 mL TRIZOL reagent (TaKaRa Bio, Shiga, Japan). cDNA was synthesized from 1µg total RNA through reverse transcription using a TaKaRa RNA PCR Kit (TaKaRa Bio). The sequences for primers are shown as following: V-ATPase (forward,5'- . Quantitative real-time PCR was performed by using 1µg of cDNA and SYBR Green (BioRad, California, USA) in 96-well plates in a LightCycler rapid thermalcycler system (MJ research, Therma). PCR products were subjected to melting curve analysis, and the data were analyzed by the 2-ΔΔCT calculation method and standardized by β-actin.

Immuno uorescent staining
Sterilized coverslips were placed in 24-well plates during GC-2 cell plating.Then, cells were washed three times with wash buffer (Immunol Fluorence Staining Kit, Beyotime). Nonspeci c binding sites were blocked for 60 min at room temperature with con ning liquid. Then, without further washing, cells were incubated with aTFEB Rabbit Polyclonal Antibody(1:200 dilution, AF8130, Beyotime) for 60 min at room temperature. TFEB staining was revealed by incubation with Alexa Fluor™ 488 donkey anti-rabbit IgG(H + L) (1:500 dilution, A21206, Invitrogen) for 60 min at room temperature. Cell nuclei were stained with DAPI (C1005, Beyotime) for 5 min at room temperature. Then, the cells were observed under laser scanning confocal microscope (IX81, Olympus, Tokyo, Japan) in random microscopic elds at ×400 magni cation.

Statistical analysis
All data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using the SPSS 20.0 statistical software program. Statistical signi cance was evaluated by one-way analysis of variance (ANOVA), followed by the least signi cant difference (LSD) test.

Hypoxia down-regulated testis V-ATPase and induced spermatogenesis reduction in mice
In our previous studies, we demonstrated that hypoxia cut off the autophagic ux of GC-2 cells in vitro by inhibiting V-ATPase; activation of autophagy can signi cantly rescue hypoxic spermatogenesis reduction (HSR) [20]. In this study, we observed that the mice exposed to hypoxia showed a considerable increase inapoptosis of spermatogenic cells ( Figure S1A). The apoptosis analysis of spermatogeniccells in seminiferoustubules by confocal microscopy showed a11-fold of apoptosisincrease in mice exposed to hypoxia for 15 din compared with the control( Figure S1B).Signi cantly decreased sperm count was also observedafter 15 days of hypoxia exposure (Fig. 1A). Meanwhile, hypoxia signi cantly increased the protein level of DR 5 (Death Receptor 5, a key molecule of death receptor apoptosis pathway) and decreased V-ATPase expression in mice (Fig. 1B). These data indicated that hypoxic exposure can triggerspermatogenesis reduction and bring about DR5 increase and V-ATPase reduction.

Overexpression of V-ATPaseimprovedspermatogenesis by inhibiting apoptosis of spermatogenic cells in hypoxic mice
Subsequently, we exploredwhether overexpression V-ATPase affected spermatogenesis under different hypoxic exposure conditions. V-ATPase KO mice was used as the positive control to further examinethe pathological damage of seminiferous tubules in LV-V-ATPase mice under different hypoxic condition. The results showed that the damage degree of seminiferous tubules in LV-V-ATPase mice was further alleviated, including the decrease of spermatogenic epithelial exfoliation and spermatogenic cell apoptosis. In contrast, V-ATPase-KO mice showed a considerable increase inapoptosis of spermatogenic cells ( Figure S2A). TUNEL assay indicated that the apoptotic rate of spermatogenic cells was remarkedly reduced after lentivirus treatment( Figure S2B). Moreover,sperm count was signi cantly increased in LV-V-ATPase group compared with the other groups( Fig. 2A). In addition, overexpression of V-ATPase by lentivirus resulted in marked reduction of DR5 levels and caspase-8 protein expression in mice under hypoxia exposure (Fig. 2B), indicating that V-ATPase may be involved in regulating the apoptosis of spermatogenic cells under hypoxia.However, it is not clear which type of spermatogenic cells apoptosis is regulated by V-ATPase.

Hypoxia down-regulated V-ATPase through inhibiting TFEB transcription and nuclear translocation in GC-2 cells
Previous reports have indicated that spermatocytes are most sensitive to hypoxia in spermatogenic cells [4]. Herein, our further experiments were conducted to determine the effect of different hypoxia time on the apoptosis of GC-2 cells by measuring the apoptotic rate at 0h, 24h and 48h after exposure at 1% O 2 . The morphology of hypoxia-treated cells was evaluated under a laser scanning confocal microscope.The results showed that there was no difference in the percent of apoptotic cells between 0h treatment group and 24h treatment group after exposure at 1% O 2 . However, a signi cant increase inchromatin condensation and nuclear fragmentationoccurred in the GC-2 cells after 24h of hypoxia treatment ( Figure S3A), re ecting the inducing effect of hypoxica on the apoptosis of GC-2 cells. The FCM assay further con rmed this observation ( Figure S3B). Furthermore, western blot results showed that hypoxia induced an increase in the protein levels of DR 5 , Caspase-8, as well as a decrease in the mRNA and protein levels of V-ATPase (Figure S3C andS3D). These results together indicated that hypoxia inhibitsV-ATPase expression and causes spermatogenesis reduction, which is associated with extrinsic apoptosis pathway of spermatocyte.
It is well known that TFEB homologues induce the transcription of several V-ATPases in Drosophila and mammals [8,34].Therefore, it is necessary to further study whether TFEB is involved in hypoxia-induced inhibition of V-ATPase expression.Therelative abundance of endogenous TFEB in total, nuclear andcytoplasmic fractions of GC-2 cells was rstly examined. Thedata showed that in total extracts of hypoxia-treated GC-2 cells, the mRNA and protein levels of TFEB were signi cantly down-regulated after 48 hof hypoxiatreatment ( Figure S4B and S4C). Importantly, 48 h of hypoxia resulted in a marked accumulation of TFEB proteinin the cytoplasmic subfraction, with a corresponding decline in thenuclear subfraction ( Figure S4D).Furthermore, immunostaining detectionrevealed that the majority of TFEB protein existed in the nucleus after 6 h of hypoxia treatment, whereas it was mainly in the cytosol after 48 h of hypoxia treatment ( Figure S4A). Then,we used GV492 particles that encodetheUbi promoterdrivenconstruct to transduce TFEB into the GC-2 cells under hypoxia exposure. Immuno uorescence, immunoblot and RT-qPCRanalysis con rmedthat the endogenousTFEB expression in the GC-2 cells was speci cally up-regulated comparedwith thoseGFP-tagged Lenti-NC vector, especiallyin the hypoxia-treated group( Figure 3A, 3B and 3C). Overexpression of TFEB signi cantly reversed the decrease in the transcriptional and protein levels of V-ATPase after 48 h of hypoxia exposure (Figure 3Band3C). Thesedata indicated that prolonghypoxia caused the deactivation of TFEB, especially in GC-2 cells, as indicated by the decreasedexpression and translocation to nuclei.TFEB overexpression resulted in an evident increase in the expression of V-ATPase.

V-ATPase de ciency induced the apoptosis of GC-2 cells via DR5 signaling
We have previously found that hypoxia can enhancethe apoptosis of GC-2 cells by inhibitingV-ATPase [20]. However, whether V-ATPase was involved in regulating DR 5 (Death Receptor 5, a key molecule of extrinsic apoptosis pathway) remained unclear. To this end, we knocked down the expression of endogenous V-ATPaseby using siRNAin order to detect the expression of extrinsic apoptosis-related markers in GC-2 cells. The results of FCM analysis showed that hypoxia alone induced 15.75%of apoptosis, whereashypoxia combinedwithsiV-ATPase augmented apoptosis to 27.15% (Fig. 4A).
Furthermore, there was a signi cant increase in the mRNA level of DR 5 in GC-2 cellsafter being treated with 1% oxygen for 48 h (Fig. 4B). Similarly, V-ATPase de ciency induced an increase in the levels of DR 5 and caspase-8 (Key effector of extrinsic apoptosis pathway) (Fig. 4C).These data indicated that V-ATPase de ciency enhances the extrinsic apoptosis pathway in GC-2 cells exposed to hypoxia.

Overexpression of V-ATPase ameliorated the apoptosis of GC-2 cells via DR5 signaling
We next sought to investigate the important roles of V-ATPaseaffecting the apoptosis of GC-2 cells.We overexpressed V-ATPase by lentivirus transfection and detected the apoptosis of GC-2 cells by TUNEL and FCM assay. The results showed decreased TUNEL-positive puncta in GC-2 cells were pretreated by V-ATPase overexpression (Fig. 5A). The FCM assay results wereconsistent with the results of the TUNEL staining (Fig. 5B). Meanwhile, overexpression of V-ATPase resulted in an evident reduction in the expressions of DR 5 and caspase-8 (Fig. 5C). Collectively, these results suggested that V-ATPase might be a critical negative regulator of spermatogenesis by in uencingtheextrinsic apoptosis pathway of spermatocytes.

Hypoxia-induced V-ATPase de ciency promoted GC-2 apoptosis via activation of JNK/c-Jun pathway
To further elucidate the underlying mechanism of V-ATPase de ciency facilitating the apoptosis of GC-2 cells, we performed RNA-seq on GC-2 cells by inducing V-ATPase de ciency through hypoxia exposure.Total 1084 and 1350 genes were consistently up-regulated or down-regulated in GC-2 cells with hypoxia exposure, respectively( Figure S5A). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested hypoxia triggered MAPK (mitogen-activated protein kinase) signaling pathway ( Figure S5B). A set of genes that codepositive mediators of JNK (A key member of the MAPK family) signaling pathway including MAPK8 (c-Jun N-terminal kinase 1, or JNK1), Jun (orc-Jun), HSF1, Rho, SHC4, CrkL, SOS1, Smad4, Atf2, bax and TRAF2were up-regulated consecutively ( Figure S5B). Further RT-qPCR and western blotvalidated thatphosphorylation of JNK and c-Jun expression wereincreased in GC-2 cells under hypoxia( Fig. 6A and 6B). Taken together, these ndings indicated that hypoxia induced the activation of JNK signaling.
Several studies have reported that the JNK signalling pathway is associated with the expression of DR (Death Receptor) markers and apoptosis [35,36]. To reveal the underlying mechanisms by which V-ATPase de ciency promotes apoptosis, we knocked down or overexpressed V-ATPase in GC-2 cells and analysed the protein expression of total and phosphorylated JNK and c-Jun by western blot. In V-ATPase overexpressed GC-2 cells, the expression of phosphorylated JNK and c-Jun were signi cantly decreased compared to CTL siRNA cells. However, knockdown of V-ATPase in GC-2 cells increased the phosphorylation of JNK and c-Jun ( Figure 6C). Furthermore, to explore the role of JNK activation in V-ATPase de ciency-induced apoptosis, we examined the effect of V-ATPase de ciency on the expression of DR related markers in the presence or absence of c-Jun siRNA. The decreased expression of death receptor (DR) related marker of DR5 and caspase-8 exhibited that c-Jun siRNA relieved expression of DR related marker after V-ATPase de ciency (Fig. 6D). The data indicated that the JNK signalling pathway mediated V-ATPase-regulated apoptosis in GC-2 cells. 7. JNK/c-Jun pathway de ciency attenuated spermatogenesis reduction induced by V-ATPase de ciency in mice under hypoxia condition Above ndings indicated that V-ATPase inhibits apoptosis activation in vitro via inhibiting the JNK/c-Jun pathway. Next, to examine whether depletion of c-Jun rescued spermatogenesis in vivo, we knocked down the expression of endogenous c-Jun using siRNA to investigate the damage of seminiferous tubules in mice by H&E, TUNEL and western blot assays. We found that mice transfected with the CTL siRNA demonstrated degeneration and detachment of spermatocytes. In contrast, c-Jun siRNAtransfected mice showed a considerable reduction in apoptosis of spermatocytes( Figure S6A and S6B).
Consistent with the results of TUNEL assay, analysis of sperm count showed that the number of mice receiving c-Jun siRNA was signi cantly higher than that of CTL siRNA.( Figure 7A). Meanwhile, the testis in CTL siRNA mice showed high expression of DR 5 and caspase-8, whilec-Jun depletion barely expressed DR 5 and caspase-8 in testis. Moreover, c-Jun depletion inhibited the expression of DR-related marker DR5 and caspase-8 (Fig. 7B). These data indicated that c-Jun depletion rescues spermatogenesis in testis and inhibits the expression of DR-related markers after hypoxia exposure.

Discussion
It has been reported that hypoxia induced spermatogenesis reduction caused by spermatocyte apoptosis in mice [4,5,37]. Continuous autophagic ux play a prosurvival role in spermatocyte cells by directly inhibiting apoptosis of spermatocytes [38]. Previously, we demonstrated that serious hypoxia inhibited autophagic ux by downregulating the expression of V-ATPase in GC-2 cells [20]. In the present study, we determined that V-ATPase de ciency resulted in more severe spermatogenesis de cits after hypoxia exposure in mice. These de cits were associated with exacerbation of spermatocyte apoptosis. These results indicated V-ATPase may exert a protective role against spermatogenesis reduction via the suppression of the apoptosis of spermatocyte. However, the mechanism by which V-ATPase inhibiting apoptosis of spermatocytes is still poorly understood.
So far, studies indicate that the main causeof spermatocyte apoptosis induced by V-ATPase de ciency is the generation of immature autophagosome resultsfrom the decreaseof autophagic ux under hypoxia stimulation, which in turn promotes the caspase-8 and death receptor (DR-) apoptosis pathway [20].
Here,wefoundthat the DR-apoptotic marker DR5 and effector caspase-8 accumulated with prolong hypoxia, which was consistent with gradual reduction of autolysosome marker V-ATPase. We have observed the decrease of lysosomal synthesis in GC-2 cells under hypoxia exposure, including the decrease of the expression of lysosomal marker LAMP2 and the decrease of the ratio of lysosomes to cells. Whether the decreased V-ATPase expression after hypoxia is connected with the observed lysosome biogenesisdefects remains to be determined.
Since TFEB was recently discovered as a major regulator o ysosome biogenesis as well as being a potential therapeutic target torescue myocardial ischemic injury and neurodegeneration [39,40], we are interested in its role in regulating V-ATPase and spermatocyte apoptosis.
Inthepresentstudy,itisnotedthatat 48 hafter hypoxia exposure boththemRNAandtotalproteinlevelofTFEBweresigni cantly down-regulated.Additionally,therewasmarked reduction ofTFEBproteininthenuclearsubfraction,suggestingthattherewas reduced translocationofTFEBintothenucleusfrom 24hto 48hafter hypoxia. On the contrary, we conducted LVmediated GC2-speci c overexpression of TFEB to show thatoverexpression of TFEB was effective in reversing the hypoxia-induced reduction of autolysosome marker V-ATPase.The results obtained in this study supporttheprevious suggestion that TFEB might regulate lysosomal biosynthesis.
The V-ATPase was ATP-dependent proton pumps that acidify intracellular compartments and ubiquitously expressed in mammals. The V-ATPase was extensively studied in cancer and neurodegenerative disease. Recent work has supported the concept that the V-ATPase playspro-survival role in cancers [41][42][43]. A number of studies have shown theoverexpression of V-ATPase subunits in various cancer cell lines and tumor samples, including breast, prostate, pancreatic, colorectal, liver, lung, ovarian, stomach, esophageal cancers and melanoma, where it showed thepro-tumoral activity [44][45][46][47][48][49][50][51][52]. In contrast, induction of apoptosis by V-ATPase inhibition has beenreported in both human and murine models encompassingmany tumor types.Tumor cells are more sensitive to V-ATPase-inhibitiondependent cancer cell death, and loss of V-ATPase activity reduces cancer cell growth [53]. Additionally, archazolid can induce p53 (tumor suppressor) protein levels by inhibitingV-ATPase expressionin cancer cells [54]. It has also been shown that V-ATPase inhibition differentially affects regulation of AMPK in tumor and nontumor cells, and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells [55]. Cancer-associated V-ATPase signi cantly induced the life span of protumor genic neutrophils by activating the intrinsic pathway of apoptosis [56]. ECDD-S27 retards the autophagy pathway by inhibiting the V-ATPase and restricts cancer cell survival [57]. Some study has found that intracellular acidosis caused by V-ATPase inhibition in breast cancer cells stabilizes the expression of the pro-apoptotic protein Bnip3, resulting in cell death [58]. It has also recently been demonstrated that treatment of breast cancer cells with the V-ATPase inhibitor archazolid disrupts the internalization of the transferrin receptor, leading to iron deprivation and subsequent apoptosis [59]. In our study, using V-ATPase lentivirus for the rst time as tool, we found thatV-ATPase overexpression caused the decline of apoptosis in GC-2 cells. Of interest, in contrast to GC-2 cells that deplete V-ATPase, V-ATPase overexpression induces the survival of V-ATPase overexpressed GC-2 cells. Similarly, in vitro studies revealed that V-ATPase reduces the apoptosis of non-cancer cells, including rat proximal tubular cells, neuronal cell and osteoclast [60][61][62]. Genetic analysis of individuals with X-linked Parkinson Disease with Spasticity (XPDS) yielded a novel candidate gene locus on the X chromosome, and it was later shown that a point mutation (c.345C > T) in exon 4 of the ATP6AP2 gene causes altered splicing of ATP6AP2 in XPDS [63]. It has been reported that in Cln1-/-mice, palmitoyl-protein thioesterase-1 (PPT1)adversely affectedV-ATPase function and dysregulated lysosomal acidi cation in other lysosomal storage disorders (LSDs) and common neurodegenerative diseases [64].Of note, we used GC-2 cells with V-ATPase silencing or overexpressed under hypoxia treatment to show for the rst time that V-ATPase in GC-2 cells regulates apoptosis of spermatocyte by acting on DR-apoptosis pathway.
It has been found that V-ATPase wasclosely associated with multiple signal transduction signaling, such as m-TOR (mammalian Target OfRapamycin), Wnt, TGF-β, Notch,G protein-coupled receptors (GPCRs) and receptortyrosine kinases (RTKs), etc. Several critical pathways of growth,survival and differentiation that are frequently altered incancers rely on the V-ATPase [65]. In Notch signaling, the Notch receptoriscleaved in Golgi and translocated to the plasma membrane, where further cleavage of the receptor occurs in response to Notch ligand binding. Cleaved Notch intracellular domain is translocated to nucleus and activates Notch target genes [66]. TGF-βprotein is glycosylated in theGolgi to form mature TGF-βand secreted into the extracellular space.TGF-βbinds to its receptor (TGF-βR)andleads to the endocytosis andphosphorylation of Smad2, which in turn activates TGF-βtarget genes [67]. During canonical Wnt signaling, the binding of ligands to the Wntreceptor complex inhibits the phosphorylation of β-catenin by GSK-3β and directs the translocation ofβ-catenin into thenucleus, where it activates the transcription of target genes Cyclin D1 and oncogene c-Myc [68]. In addition, it was found that V-ATPase is critical for sensing of amino acids and subsequent activation of mTOR complex 1 (mTORC1). Amino acids stimulate recruitment of mTORC1 to the lysosomal surface, where its direct activator,Ragulator(a family of four GTPases that are related to Ras, RagGTPases) was associatedtightly with the V-ATPase [69,70]. were markedly promotedby V-ATPase de ciency, which was consistent withtheactivation of JNK signaling in glial cells, monocyte-derived macrophages and RAW 264 [71][72][73]. Moreover, some other studies have shown that V-ATPase inhibits JNK pathway in drosophila epithelium, mouse bone marrow macrophages and osteoclast [74][75][76]. The exact mechanism by which V-ATPase acts on glycosylation, MMPs, γ-secretase, endocytosis and degradation in V-ATPase-mediated JNK signaling still needs further investigation.
The JNK, as a MAPK member, regulates various cell functions, such as proliferation, apoptosis, and differentiation [77]. The mechanism underlying JNK-regulated DR-apoptotic pathway has been largely investigated in cancer cells. It has been demonstrated that erinacine A induces apoptosis by acting JNK, p300, and NFκBp50signaling molecules and an increasingthe cellular transcriptional levels of TNFR, Fas, and FasL [78]. The attenuation of ERK1/2 phosphorylation accompanied by the activation of JNK was detected in D. bulbifera ethyl acetate fraction (DBEAF)-induced activation of death receptor, Fas and HCT116 cell apoptosis [79]. The capsular polysaccharide induced extrinsic cell apoptosis by upregulating FAS/FASL signaling proteins and cleaved-caspase-8 and promoted a ROS-dependent intrinsic cell apoptosis by activating a JNK and p38 signaling but not ERK1/2 signaling of mitogen-activated protein kinases (MAPK) pathways [80]. The JNK inhibition was validated to block irradiation-induced FasL expression, which was critical in determining non-irradiated hepatocyte injury [81].Short hairpin RNA (shRNA)-mediated knockdown of JNK con rmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited signi cantly reduced TRAIL/sunitinibmediatedcolon cancer apoptosis [82]. Subcutaneous tumor growth analysis revealed that Mucosaassociated lymphoma antigen 1 (a lymphoma oncogene, MALT1) gene silencing signi cantly increased melanoma apoptosis and forced expression of the c-Jun upstream activator MKK7 [83]. Quercetin activated c-Jun N-terminal kinase (JNK) in a dose-dependent manner, which in turn induced the proteasomal degradation of cFLIP, and JNK activation also sensitized pancreatic cancer cells to TRAILinduced apoptosis [84].Tanshinone IIA (antitumor drug, TIIA) promoted JNK-mediated signaling to upregulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5 [36].Saikosaponin D (antitumor drug) alone or in combination with SP600125 (JNK inhibitor) activated caspase-3, -8 in human osteosarcoma U2 cells [85]. Inthe present study,the activation of JNK/c-Jun in spermatocyte was enhanced by V-ATPase de ciency in vitro, while inhibitionof JNK phosphorylation alleviated spermatocyte apoptosis, therebyindicating that V-ATPase attenuates spermatocyte apoptosis,at least in part, via the suppression of the JNK/c-Jun pathwayafter hypoxia exposure.
Emerging studies demonstrated thatJNK signaling regulates non-cancer cell apoptosis induced by ischemic hypoxia, including neuronal, astrocyte and cardiomyocyte [86][87][88]. Various JNK-related synthetic inhibitors have been reported in ischemic hypoxia injury, such as micromolecules SP600125 and IQ-1S. It has been reported that SP600125 treatment inhibits JNK activation and provides neuroprotection in ischemia/reperfusion via inhibiting neuronal apoptosis [89]. IQ-1S releases nitric oxide in the course of redox biological transformation process and improves the results of stroke in a cerebral reperfusion mouse model [90]. In this study, our data provides evidence that V-ATPase de ciency leads to increased phosphorylation of JNK/c-Jun in GC-2 cells under hypoxia exposure. In addition, the V-ATPase de ciency-induced phosphorylation of JNK/c-Jun and the cell apoptosis activity was reduced when c-Jun was inhibited by RNA interference (RNAi). Our data suggest that JNK activation might be a key event in V-ATPase-mediated apoptosis in GC-2 cells.
In conclusion, V-ATPase de ciency aggravates spermatogenesis de cits under hypoxia exposure, which may be due to the exacerbation ofspermatocyte apoptosis. The aggravated spermatocyte damage is associated withenhanced DR-apoptotic pathway activation, which is mediated by V-ATPase via the JNK/c-Jun signal. These results demonstrate the protective role of the V-ATPase against spermatocyteinjury andprovide evidence for the exploration of V-ATPase-based treatmentsfor hypoxiainduced spermatogenesis reduction (Figure 8).

Consent for publication:
The participant has consented to the submission of the case report to the journal. The authors declare that they have no con ict of interest.
Availability of data and material: h ttps://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA608077 Funding: This work was supported by the National Natural Science Foundation of China (81701855) Authors' contributions: