Abstract graphic file with name se3c00068_0008.jpg To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm–hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction. Keywords: G-quadruplexes, mitochondria, spermatozoa, fertilization failure, mitophagy __________________________________________________________________ Infertility is a major global health problem, affecting 8–12% of couples in the reproductive age group, among whom approximately 50% are infertile due to male factors.^[50]1 Male infertility prevalence increased by 0.291% per year between 1990 and 2017.^[51]2 The World Health Organization (WHO) recommends routine semen analysis as the first step in assessing male fertility potential.^[52]3 However, reference indicators from semen analysis have limited accuracy for determining male reproductive potential or predicting reproductive success.^[53]4 Among the known factors, most male infertility cases are idiopathic.^[54]5 Repeated fertilization failure refers to the inability to obtain embryos or poor embryo development quality after IVF. At present, fertility outcome potential is normally judged merely according to the routine sperm parameters, with unexplained fertilization failure an ongoing problem. Nor can current detection indicators provide effective or feasible improvement and treatment planning. Therefore, there is still a lack of clinical etiological indicators for sperm samples in male patients with unexplained infertility. The flagellum of a mature spermatozoon is the major region controlling sperm motility, fertilization, and other functions. It consists mainly of a midpiece with mitochondrial sheath, a principle piece, and an end tail.^[55]6 Mitochondrial defects are closely linked to male fertility since ATP, the major energy product necessary for sperm functions, is produced by the mitochondrial sheath.^[56]7 Spermatozoa ATP is used not only for sperm motility but also for capacitation, acrosome reaction, hyperactivation, and oocyte penetration.^[57]8 Spermatozoa can utilize both oxidative phosphorylation (OXPHOS) and anaerobic glycolysis.^[58]9 Mitochondria possess their own genome, called mitochondrial DNA (mtDNA). It is generally accepted that, as the proportion of abnormal mtDNA increases beyond a certain level, cellular dysfunction and symptom manifestation are caused by decreased energy production.^[59]8 Similarly, mtDNA quantity in spermatozoa is directly correlated to sperm quality. Selective autophagy of mitochondria, known as mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria in sperm.^[60]10 It is widely accepted that mtDNA is maternally transmitted to the offspring and that paternal mitochondria and their genome are generally eliminated in the embryo by an autophagy-related degradation mechanism, usually referred to as mitochondria autophagy.^[61]11 Thus, mitochondria are believed to play key roles in sperm motility, capacitation, and fertilization capability processes, highlighting their importance in male infertility.^[62]12 G-Quadruplexes (G4s) are the noncanonical four-stranded nucleic structures folded by DNA or RNA sequences particularly abundant in adjacent guanines.^[63]13 G4s have been implicated in multiple cellular functions, including chromatin epigenetic regulation, DNA recombination, transcriptional regulation of gene promoters and enhancers, and translation.^[64]14 G4s are also a potential therapeutic target and have been closely related to neurological diseases,^[65]15 cancer therapy,^[66]16 and embryonic development.^[67]17 A previous study showed that G4s also exist in mitochondria and that they may cause mitochondrial genome instability and thus induce mitochondrial dysfunctions.^[68]18 However, questions about mtDNA G4s and their roles in human spermatozoa remain. To find a novel etiological indicator and explore the effects of mtDNA G4s in spermatozoa, we designed a fluorescent probe targeting mtDNA G4s in the previous study, which has been used for simple and rapid tracking (<10 min) in tumor cells.^[69]19 After verifying the high sensitivity and specificity of the probe in spermatozoa, we further detected the level of mtDNA G4s in the spermatozoa with multiple clinical insemination failures and investigated whether it could be restored by a mitophagy inducer ([70]Figure [71]1 ). The goal of this study was to shed some light on the effects of mtDNA G4s on certain pathological states of spermatozoa, to be used as a potential novel etiological indicator for male infertility. Importantly, we dissected sperm mitochondrial G4s structures using the novel fluorescent probe as a new etiological biomarker for patients with clinical infertility and developed a new potential treatment for patients with mtDNA G4 dysfunction. Figure 1. [72]Figure 1 [73]Open in a new tab Study flowchart. TPE-mTO can be used for simple and rapid spermatozoa tracking by mixing with sperm and testing directly by flow cytometry. Failed fertilization caused by increased mtDNA G4s can be rescued by the mitophagy inducer. Methods Participants The Ethics Committees of the West China Second University Hospital of Sichuan University approved the study. Each participant signed informed consent. The inclusion and exclusion criteria were according to the WHO guidelines.^[74]20 Control semen samples were collected from healthy donors whose fertility had been previously confirmed. Sperm motility was analyzed using a CASA instrument (Chengdu Puhua Technology Co., Ltd, Chengdu, China). Institute of Cancer Research mice (specific pathogen-free) purchased from Chengdu Dossy Experimental Animals Co. Ltd. (Chengdu, China) were used to obtain sperm from epididymis. TPE-mTO Probe Staining Briefly, semen was washed three times with twice the volume of phosphate-buffered saline (PBS). The semen samples were then centrifuged at 300–600g for 10 min. Then, G-IVF PLUS (Vitrolife, Sweden) was used to obtain an appropriate concentration of sperm using the swim-up method. A previously synthesized G4 probe, TPE-mTO, was added to sperm at different concentrations and incubated at room temperature for 10 min. The prepared costained sperm (without any washing steps) that was counterstained with Hoechst33342 (Sigma-Aldrich) to label the nuclei was placed on NEST 3.5 mm glass-bottom dishes coated with polylysine (Solarbio, P2100, China) to obtain images using FV1000 (Olympus, Japan) and LSM780 (Zeiss, Germany) laser confocal microscopes. TPE-mTO probe staining was also analyzed using image-based flow cytometry (IBFC) (Amnis, Germany). TPE-mTO was excited at 488 nm. Drug Treatments and Sperm Transfection All drugs were prepared fresh with dimethyl sulfoxide (DMSO) to the desired concentrations before each experiment. The prepared stock solution was stored at −20 °C. All drugs were purchased from Sigma-Aldrich. The final disposal conditions for all drugs were as follows: PDS, 0–10 μM, 1 h; tmpyp4.0–10 μM, 1 h; paclitaxel, 10 μM, 4 h; amitriptyline, 10 μM, 4 h; mirtazapine, 10 μM, 4 h; and H[2]O[2], 30%, 30 min. The negative control group was treated with 0.05% DMSO. CM22 and DS26 were synthesized from Tsingke Biotechnology Co., Ltd. (Chengdu, China) and transfected into spermatozoa with jetPRIME (Polyplus-transfection, 101000046, Germany). The sequence used was as follows: * CM22, 5′ TGAGGGTGGGTAGGGTGGGTAA 3′ * DS26, 5′ CAATCGGATCGAATTCGATCCGATTG 3′ All drug-treated spermatozoa underwent a one-step wash with PBS to remove the drug-treated buffer before TPE-mTO probe staining. Cell Culture HepG2, A549, and SKOV-3 were obtained from the American Type Culture Collection. HepG2 cells were grown in DMEM supplemented with 10% FBS. A549 cells were cultured in RPMI 1640 medium (90% 1640 medium + 10% FBS). The SKOV-3 cells were expanded in RPMI 1640 medium containing 10% FBS. The final disposal conditions for cells were as follows: PDS, 1 μM, 18 h and Tmpyp4, 2 μM, 18 h. Human Sperm Penetration of Zona-Free Hamster Egg Assay Detection Hamster oocytes were obtained by superovulation.^[75]21 Human sperm penetration of the zona-free hamster egg assay (SPA) was used as described previously to evaluate sperm fertilizing ability.^[76]21 Washed spermatozoa were preincubated in human tubal fluid (HTF) medium with or without 3 μM PDS for 5 h under capacitating conditions. Zona-free hamster eggs were added into sperm suspensions (50:50 μL, eggs: sperm ≥ 1.5:1000) and co-incubated for 4 h in the HTF medium with or without 10 μM INK-128. After co-incubation, eggs were washed three to four times with HTF medium without any drugs and transferred to a slide, the corners of which were coated with drops of a paraffin wax–vaseline mixture (1:9, V/V) and compressed under a glass coverslip. The eggs were examined by phase-contrast microscopy (×40). The decondensed head and tail of sperm were presented in the ooplasm as evidence of fertilization. Finally, the penetration rate of sperm per egg was calculated as the sperm fertilization capacity.^[77]21 Mitochondrial Membrane Potential Detection Semen was washed with PBS three times and treated with or without 3 μM PDS for 1 h. The mitochondrial membrane potential of treated sperm were measured by JC-1 dye using the JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, C2006, China) and flow cytometry (Beckman, American). JC-1 dyes were excited at 488 nm and detected at 535 nm emission for JC-1 monomers and excited at 550 nm and detected at 600 nm emission for JC-1 aggregates. Mitochondrial Reactive Oxygen Species-Level Detection Semen was washed with PBS three times and treated with or without different PDS concentrations for 1 h. Mitochondrial reactive oxygen species (ROS) levels of spermatozoa were determined with a sperm reactive oxygen species staining kit (DCFH-DA/MitoSOX Red flow cytometry) (PH70922112701). DCFH-DA/MitoSOX Red flow fluorescence was measured using flow cytometry (Beckman, American). Oxygen Consumption Rate Detection A Seahorse XFe Extracellular Flux Analyzer (Seahorse Bioscience) was used to determine the oxygen consumption rate (OCR, pmol/min) of treated sperm according to the manufacturer instructions. In eight-well Seahorse test plates with polylysine, sperm were plated at a density of 50,000 cells per well (Solarbio, P2100, China). Spermatozoa with or without 3 M PDS were cultured for 1 h in a 37 °C incubator without CO[2]before the start of each experiment. The medium was then changed with XF assay media, which contains unbuffered DMEM, 5.5 mM glucose, and 0.5 mM carnitine. Immunofluorescence Four percent paraformaldehyde was used to fix sperm samples onto slides for 10 min and PBS-washed three times. The slides were permeabilized with 0.3% Triton X-100 and blocked with 5% bovine serum albumin in PBS. The corresponding primary antibodies (anti-VCP [1:100] [ab109240; abcam]) were incubated with the slides at 4 °C overnight. The next day, the slides were washed with PBS and incubated with Alexa Fluor 594 (1:1000) (A11005; Thermo Fisher)-labeled secondary antibodies at room temperature for 1 h. The nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). A laser scanning confocal microscope (Olympus, Japan) was used to obtain images. One hour prior to the beginning of immunofluorescence experiments, the sperm were washed and treated with or without 3 μM PDS for 1 h. RNA-seq Following the manufacturer’s instructions, total RNA was extracted using a TRIzol reagent (Thermo Fisher, 15596018). High-quality RNA samples with integrity numbers >7.0 were used to generate sequencing libraries after the total RNA quantity and purity were determined using a Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit (Agilent, CA, 5067-1511). Using Dynabeads Oligo (dT) (Thermo Fisher, CA), mRNA was isolated from total RNA (5 ug) after it had been extracted by two rounds of purification. Following purification, using a divalent cation and high temperature (Magnesium RNA Fragmentation Module [NEB, cat. e6150] for 5–7 min), the mRNA was broken into small pieces. Then SuperScript II Reverse Transcriptase (Invitrogen, cat.1896649) was used to create the cDNA by reverse-transcribing the cleaved RNA fragments. U-labeled second-stranded DNAs were then synthesized with E. coli DNA polymerase I (NEB, cat.m0209), RNase H (NEB, cat.m0297), and dUTP solution (Thermo Fisher, cat. R0133). The blunt ends of each strand were then given an A-base to help ligate them to the indexed adapters. Each adapter has a T-base overhang for attaching to the DNA fragments with their A-tailed ends. The fragments were ligated to dual-index adapters, and AMPureXP beads were used for size selection. The U-labeled second-stranded DNAs were treated with the heat-labile UDG enzyme (NEB, cat. m0280) before the ligated products were amplified with a polymerase chain reaction (PCR) under the following conditions: initial denaturation at 95 °C for 3 min, 8 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, and final extension at 72 °C. The resultant cDNA collection had an average insert size of 30050 bp. Finally, the vendor’s suggested technique was followed to complete the 2150 bp paired-end sequencing (PE150) on an Illumina NovaseqTM 6000. Using the R tool DESeq. 2, differentially expressed genes at the transcription level were analyzed (1.32.0).^[78]22 Reverse Transcription Quantitative PCR The TRIzol (Invitrogen) reagent was used to extract total RNA from mouse tissues or cells and the RevertAid First-Strand cDNA Synthesis Kit to produce cDNA (Thermo Fisher, Waltham). On an iCycler reverse transcription (RT)-PCR detection system, real-time PCR was carried out using SYBR Premix Ex Taq II (TaKaRa, Dalian, China) (Bio-Rad Laboratories, Hercules). Data analysis employed the cycle threshold (CT) technique. For every sample, each test was carried out in triplicate. The actin gene served as an internal control. [79]Supplementary Table SII contains a list of the primers for real-time PCR. Protein-seq A nano UPLC (EASY-nLC1200) connected to a Q Exactive HFX Orbitrap instrument with a nanoelectrospray ion source (Thermo Fisher Scientific) was used to separate and analyze 2 g of total peptides for each sample. A reversed-phase column (Reprosil-Pur 120 C18-AQ, 100 μm ID × 15 cm, 1.9 m, Dr. Maisch) was used for separation. Phase A was H[2]O with 0.1% FA and 2% ACN; phase B was 80% ACN and 0.1% FA as mobile phases. Sample separation used a gradient for 120 min at a flow rate of 300 nL/min. Gradient B: 2% for 2 min, 5% for 88 min, 22% for 26 min, 45% for 2 min, and 9% for 2 min. Using an Orbitrap analyzer in profile and positive mode, data-dependent acquisition (DDA) was carried out on MS1 and MS2 with resolutions of 120,000 (@200m/z) and 15,000 (@dynamic first mass), respectively. AGC targets for MS1 and MS2 were set to 3E6 and 50 ms and 1E5 and 110 ms, respectively. The top 20 most intense ions were fragmented by HCD using an isolation window of 1.2m/z and a normalized collision energy of 27%. Single charged peaks and peaks with a charge >6 were excluded from the DDA method, and the dynamic exclusion time frame was 45 s. Proteome Discoverer software (Version 2.4.0.305) and the integrated Sequest HT search engine were used to process the vendor’s raw MS files. The UniProt-Homo sapiens-9606-2022-11. Fasta species-level UniProt FASTA databases were searched using MS spectra lists, with carbamidomethyl (C) as a fixed modification, oxidation (M), and acetyl (protein Nterm) as variable modifications. As proteases, trypsin was employed. There may have been a maximum of two missed cleavages. The PSM and peptide levels both had a 0.01 false discovery rate. With an initial precursor mass fluctuation of up to 10 ppm and a fragment mass divergence of 0.02 Da, peptide identification was carried out. For protein measurement and normalization, the total peptide quantity and Razor peptide were employed. Default values for all other parameters were set. Statistical Analysis All data are expressed as mean ± standard deviation (SD). GraphPad Prism software (version 8.00) was used to analyze all statistics. Two-tailed Student’s t-tests were used for between-group comparisons. One-way analysis of variance was used to compare more than two groups, and Dunnett’s test was used to compare between groups. Significant between- and among-group differences are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001. Results and Discussion mtDNA G4-Specific Fluorescent Probe TPE-mTO Monitoring mtDNA G4s in Spermatozoa We previously^[80]19 constructed a novel mtDNA G4-specific fluorescent probe TPE-mTO ([81]Figure [82]1 ). Since we found that TPE-mTO probes stain the nucleus of fixed cells according to the experimental conditions of previous studies^[83]19 ([84]Figure S1), to ensure the specificity of staining, all stained spermatozoa herein were screened by the swim-up method to ensure their viability and survival rate and then used imaging flow (IBFC) to determine the optimal staining concentration of the probe. The optimal concentration of TPE-mTO to specifically detect mtDNA G4s in spermatozoa was 6.25 μM with specific mitochondrial localization ([85]Figure S2). When incubated at 6.25 μM for 10 min, sperm motility was unaffected by TPE-mTO, although curvilinear velocity (VCL) decreased significantly compared with the control group ([86]Figure S3), suggesting that the probe had no effect on sperm motility (although it had a slight effect on sperm trajectory under this condition). To further verify probe sensitivity and specificity, we employed pyridostatin (PDS) and Tmpyp4 to regulate the folding of mtDNA G4 structures. PDS and Tmpyp4 can promote the folding of telomeric G4s.^[87]23 We verified that there is little competition between PDS/Tmpyp4 and TPE-mTO probes ([88]Figure S4). The proportion of positive cells significantly increased after PDS and Tmpyp4 treatment of spermatozoa ([89]Figure [90]2A,B) as detected by IBFC. A remarkable increase in fluorescence intensity on mitochondria in individual spermatozoon was also observed ([91]Figure [92]2C). We previously found that TPE-mTO could bind CM22 (G4s) and lead to strong fluorescence in vitro.^[93]24 Here, transferring CM22 into spermatozoa also led to a detectable fluorescence enhancement ([94]Figure [95]2D), further suggesting that TPE-mTO was mainly bound to G4s. DS26, a dsDNA structure, was used as a negative control. Similarly, the fluorescence enhancement was also found in mouse spermatozoa with CM22 transfection ([96]Figure S5). These results indicated that the TPE-mTO probe could monitor mtDNA G4 in human and mouse spermatozoa sensitively and specifically. The probe could also achieve fluorescence for in vivo diagnosis of sperm with only two steps of dyeing and machine detection. Figure 2. [97]Figure 2 [98]Open in a new tab Evaluation of TPE-mTO as a mtDNA G4-specific fluorescent probe in spermatozoa. (A,C) IBFC analysis and fluorescence images of spermatozoa stained with TPE-mTO (6.25 μM, λex = 488 nm, channel 2) in PBS buffer, after spermatozoa were treated with 3 μM PDS for 1 h and 5 μM Tmpyp4 for 1 h. Scale bars for spermatozoa image = 20 μm. (B) Statistical map of group differences in positive cells between control and spermatozoa with PDS and Tmpyp4 treatments at a threshold of p ≤ 0.05, n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Confocal fluorescence images (Olympus, FV1000) of spermatozoa with TPE-mTO (6.25 μM, λex = 488 nm, red) under different conditions that spermatozoa were transfected with CM22 (parallel G4s oligonucleotides) and DS26 (double-strand DNA oligonucleotides) in PBS buffer (contain 50 mM K+). CM22 and DS26 were previously transfected into human sperm using jetPRIME. Scale bars for spermatozoa image = 10 μm. The nucleus is indicated by Hoechst33342 (blue). mtDNA G4s as a Potential Indicator of Spermatozoa-Related Etiology in Fertilization Failure and Low Embryo Quality To explore the relations between the mtDNA G4s and spermatozoa, sperm were treated with chemicals that have been reported to cause negative effects and lead to pathological damage (e.g., DNA damage, respiratory depression, oxidative stress injury). Paclitaxel induces DNA damage in sperm.^[99]25 Amitriptyline and mirtazapine act generally as inhibitors of complex I and complex IV of the electron transport chain.^[100]26 H[2]O[2] is the most commonly used inducer of oxidative stress injury in vitro.^[101]27 The IBFC results indicated that the proportion of mtDNA G4 positive spermatozoa with respiratory chain inhibition and oxidative stress injury increased significantly by TPE-mTO probe monitoring, while the amount of mtDNA G4s in spermatozoa with DNA damage was negligible ([102]Figure S6). Meanwhile, 18 spermatozoa samples from patients with multiple clinical insemination failures were collected ([103]Table S1); their routine sperm tests were normal ([104]Table [105]1 ) and female infertility factors had been excluded. After detecting mtDNA G4s by the TPE-mTO probe, IBFC results indicated that the proportion of mtDNA G4 positive spermatozoa in semen with failed fertilization was significantly increased ([106]Figure [107]3A,B), and the fluorescence of mitochondria in single spermatozoon was enhanced ([108]Figure [109]3C). The area under the curve (AUC) of the receiver operating characteristic (ROC) curve was 0.9217. All of the results suggested that increased mtDNA G4s were related with pathological spermatozoon, which further indicated that mtDNA G4s can be used as a potential indicator of spermatozoa etiology in fertilization failure and poor embryo development. Table 1. Medical Chart Information Regarding Semen Collected from Patients with Multiple Clinical Insemination Failures. patient count (×10^6) motility (%) volume (mL) pH density (×10^6/mL) liquefaction time 1 81.2 92 3.9 7.4 20.82 <30 min 2 129 78 3.3 7.4 39.09 3 120.6 71 4.1 7.4 29.41 4 207.2 74 1(↓) 7.4 207.20 5 117 72 4.7 7.4 24.89 6 54.9 81 2.6 7.4 21.12 7 62 67 3.1 7.4 20.00 8 54 88 2.6 7.4 20.77 9 118.18 35 3.8 7.8 31.10 10 194.62 42 3.7 7.8 52.60 11 71.2 90 4 7.4 17.80 12 73.9 95 5 7.4 14.78 13 182 93 2.4 7.4 75.83 14 112.9 87 2.6 7.4 43.42 15 73 75 3.1 7.4 23.55 16 104 90 3.4 7.4 30.59 17 43 76 2.7 7.4 15.93 18 72 65 4.3 7.4 16.74 normal value >15 >32 >1.5 7.2–7.8 >15 ≤30 min [110]Open in a new tab Figure 3. [111]Figure 3 [112]Open in a new tab Exploration of the relations between mtDNA G4s and pathological spermatozoa. (A,C) IBFC analysis and fluorescence images of normal spermatozoa and failed fertilization spermatozoa stained with TPE-mTO (6.25 μM, λex = 488 nm, channel 2) in PBS buffer. Scale bars for spermatozoa image = 20 μm. (B) Statistical map of group differences in median fluorescence intensity between controls and patients with multiple clinical insemination failures at a threshold of p ≤ 0.05, n = 22 (control) and 18 (patients), *p < 0.05, **p < 0.01, and ***p < 0.001. (D) ROC curve of the increased mtDNA G4s based on the median fluorescence intensity. The 95% confidence interval of the AUC was estimated by the Delong method. The novel biomarker mtDNA G4s have high predictive capacity for fertilization failure, which cannot be replaced by other indicators like DFI and oxidative stress. This detection technology could also achieve noninvasive fluorescence imaging of sperm in vivo, which could be detected after the sperm were incubated with the probe for 10 min without the need for repeated washing. This method is thus efficient and convenient for clinical detection. Increased mtDNA G4s Can Cause Mitochondrial Damage and Further Abnormal Fertilization Capacity Which Can Be Rescued by an Autophagy Inducer To investigate the effects of increased mtDNA G4s on fertilization ability, PDS was used to increase the amount of mtDNA G4s in spermatozoa. To mimic the elevated mtDNA G4s in patients with multiple clinical insemination failures, we used IBFC to examine the mtDNA G4s in sperm treated with various PDS concentrations to find that 3 μM was the optimal concentration ([113]Figure S7). CASA analysis indicated that the motility of spermatozoa treated with PDS was normal ([114]Figure S8A). The beat cross-frequency and straightness were slightly reduced in spermatozoa with PDS ([115]Figure S8B,C). There were no significant differences in spermatozoa with PDS on parameters including average path velocity, straight line velocity, VCL, linearity, and amplitude of lateral head displacement ([116]Figure S8D–H). These cumulative results suggest that PDS had no significant effects on sperm movement. The results of human sperm penetration of zona-free hamster egg assay (SPA) suggested that the penetration rate per egg by spermatozoa was reduced significantly (by 50%) by PDS treatment ([117]Figure S8I), indicating that increased mtDNA G4s may reduce fertilization ability, while its effect on sperm movement is negligible. Detection of the mitochondrial membrane potential by the cationic dye JC-1 in spermatozoa showed that the mitochondrial membrane potential of spermatozoa with increased mtDNA G4s was reduced ([118]Figure S9A,B). Mitochondrial ROS levels were measured using DCFH-DA/MitoSOX Red staining. There was little effect on mitochondrial ROS levels of spermatozoa with PDS/Tmpyp4 treatment ([119]Figure S9C,D). These results suggest that increased mtDNA G4s causes mitochondrial damage without much effect on the mitochondrial ROS production in our experimental setting. Valosin-containing protein (VCP) is a key protein involved in mitophagy.^[120]28 RT-PCR ([121]Figure [122]4 A) and immunofluorescence ([123]Figure [124]4B,C) results revealed that VCP expression in the PDS group was downregulated, indicating that increased mtDNA G4s may lead to abnormal mitochondrial clearance during fertilization and then to failed fertilization. Figure 4. [125]Figure 4 [126]Open in a new tab Induction of abnormal mitophagy can restore abnormal fertility caused by increased mtDNA G4s. (A) RT-PCR revealed the quantified mRNA expression level of VCP normalized to ACTIN mRNA after spermatozoa were treated with 3 μM PDS for 1 h. (B) VCP/DAPI ratio of immunofluorescence of spermatozoa with 1 h treatment of 0 μM and 3 μM PDS. The sperm count is assured to exceed 1000. Immunofluorescence revealed the protein expression level of VCP under different conditions after spermatozoa were treated with 3 μM PDS for 1 h. (C) Immunofluorescence revealed the protein expression level of VCP under different conditions after spermatozoa were treated with 3 μM PDS for 1 h. The VCP/DAPI ratio of immunofluorescence of spermatozoa with 1 h treatment of 0 and 3 μM PDS. (D) SPA analysis of average number of sperm per egg under different conditions after spermatozoa were treated with 3 μM PDS for 5 h and 10 μM INK-128 for 4 h. All data are presented as mean ± SD from three independent experiments, and statistical significance was determined by t-test as (NS) not significant, *p < 0.05, **p < 0.01, or ***p < 0.001. INK-128, an inhibitor of the mechanistic target of rapamycin, can promote mitophagy.^[127]29 After INK-128 treatment of spermatozoa with increased mtDNA G4s, SPA results showed recovery of the penetration rate per egg by spermatozoa ([128]Figure [129]4 D), indicating that sperm with abnormal fertilization ability due to increased mtDNA G4s can be restored by enhancing mitophagy. These results illustrate that increased mtDNA G4s may cause mitochondria damage and lead to further abnormal fertilization ability, which can be reversed by promoting mitophagy. These results indicate that paternal mitochondria are removed during fertilization.^[130]30 All paternal mtDNAs are degraded by mitophagy during fertilization.^[131]11 DNA G4s were also reported to regulate autophagy.^[132]31 Because autophagy can be induced by some known compounds, such as INK-128, rapamycin,^[133]32 and spermidine,^[134]33 the close relations between mtDNA G4s and mitophagy in spermatozoa showed herein suggest that mtDNA G4s may be a potential therapeutic target for rescue by autophagy inducer. Consistent with this, we observed significant recovery in increased mtDNA G4 sperm with INK-128 treatment. Therefore, our study showed that an autophagy inducer is essential in the treatment of mtDNA G4-related fertilization failure. In addition, in the case of abnormal increase in mtDNA G4s during in vitro fertilization (IVF) treatment, adding autophagy inducers in the medium might be considered. This would be of great significance for clinical practice, improving IVF success rates. Proteomic and Transcriptomic Correlation Analysis Revealed Increased mtDNA G4s in Spermatozoa, Mainly Downregulated OXPHOS To further identify the key pathway by which increased mtDNA G4s causes mitochondria damage, we performed RNA-seq and proteomic on spermatozoa with 3 μM PDS treatment for 1 h. RNA-seq revealed that 22 genes were upregulated and 49 genes were downregulated ([135]Figure [136]5 A,B). The results of the RNA-seq Pearson correlation coefficient between samples indicated that the samples were well clustered ([137]Figure S10). These results indicate that the increased mtDNA G4s affected expressions of key metabolism-related genes and pathways. The Kyoto encyclopedia of genes and genomes (KEGG) pathway and gene ontology term of RNA-seq were analyzed based on gene set enrichment analysis (GSEA). Downregulated genes were similarly enriched in OXPHOS ([138]Figures [139]5C and [140]S11). The downregulation of key genes ATP5F1E, PPA2, NDUFS8, COX5B, and ATP5MC2 mRNA expression was verified in spermatozoa with PDS treatment compared with the control group ([141]Figure [142]5D). Figure 5. [143]Figure 5 [144]Open in a new tab RNA-seq indicated that promotion of mtDNA G4 folding caused downregulated OXPHOS. (A) Heatmap of differentially expressed transcripts in PDS-treated spermatozoa. Red and blue indicate whether the expression value is below (blue) or above (red) the mean expression value across samples (the data were normalized from −1 to + 1). n = 3 for each group. Padj ≤ 0.05, |log2 FC| ≥ 1. (B) Volcano map analysis of differentially expressed transcripts, showing 22 upregulated and 49 downregulated genes. Padj ≤ 0.05, |log2 FC| ≥ 1. (C) Downregulated genes were analyzed for KEGG term enrichment by GSEA, which showed that the pathways enriched in spermatozoa treated with 3 μM PDS were reduced OXPHOS. (D) OXPHOS-related gene expression. ACTIN was used as an internal reference gene for qPCR. (E) OXPHOS of spermatozoa with 1 h treatment of 3 μM PDS, as measured by OCR. The number of samples in each group was n = 3, and *p < 0.05, **p < 0.01, and ***p < 0.001. The differentially expressed proteins from proteomic analysis are shown in [145]Figure [146]6 A, of which 126 were upregulated and 30 were downregulated ([147]Figure [148]6B). The KEGG pathway of proteomic and transcriptomic correlation analyzed the differential pathway between the PDS-treated and control groups ([149]Figure [150]6C). OXPHOS was also a significant pathway. The differentially expressed proteins in the OXPHOS pathway are shown in [151]Figure [152]6D. Protein–protein interaction network analysis indicated that the differential proteins of sperm treated with PDS were mainly related to their energy metabolism, and fewer protein molecules were directly related to fertilization and sperm motility, further indicating that PDS may affect the fertilization ability of sperm by affecting their energy metabolism ([153]Figure S12). The Seahorse experiment results indicated that the OCR value of spermatozoa at the baseline decreased significantly after PDS treatment ([154]Figure [155]5E), suggesting that increased mtDNA G4s may decrease the OXPHOS in spermatozoa. That increased mtDNA G4s, mainly downregulated OXPHOS in spermatozoa, may also lead to mitochondria damage and further abnormal fertilization abilities. Figure 6. [156]Figure 6 [157]Open in a new tab Proteomic and transcriptomic correlations were used to analyze the increased mtDNA G4s, which mainly affected OXPHOS. (A) Heatmap of differentially expressed proteins in PDS-treated spermatozoa. Red and blue indicate whether the expression value is below (blue) or above (red) the mean expression value across samples (the data were normalized from −1 to + 1). n = 3 for each group. Padj ≤ 0.05, |log2 FC| ≥ 1. (B) Volcano map analysis of differentially expressed proteins displaying 126 upregulated and 30 downregulated proteins. Padj ≤ 0.05, |log2 FC| ≥ 1. (C) KEGG pathway enrichment analysis of key proteins (PDS vs CTL). (D) OXPHOS pathway map of differentially expressed proteins enriched by KEGG between the PDS and CTL groups. Bright red and blue represent upregulated and downregulated differentially expressed proteins, respectively. Conclusions Herein, we applied our previously engineered chemical probe to monitor mtDNA G4s in spermatozoa. This probe can achieve fluorescent real-time imaging of sperm, and detection can be tracked simply and rapidly. After detection, patients with fertilization failure showed increased mtDNA G4s. Increased mtDNA G4s mainly lead to reduced OXPHOS and causes mitochondria damage. Damaged mitochondria cannot be removed effectively, which leads to abnormal fertilization; this can be effectively restored by improving mitophagy. This study thus provides a novel etiological biomarker for patients with clinical infertility and a new potential treatment for those with mtDNA G4 dysfunction. Acknowledgments