Animal breeding and model construction

All of the animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals formulated and implemented by the National Institutes of Health. The animal experiments were approved by the experimental animal management and ethics committee of West China Second Hospital. All of the animals were kept in specific pathogen free (SPF) animal rooms for 12-h day–night alternation, under a standard animal diets and with free access to water. The details for generating Qrich2 KO mice were described in a previous study [25]. For comparing the effect of the diets, 12 male C57BL/6 J mice aged 4 weeks were randomly divided into two groups and fed with a control diets or Gln/Glu-free diets for 5 weeks. The specific feed ingredients are shown in Table S1.

Participants involved in the study

Eight healthy Han males with normal fertility and five patients clinically diagnosed with asthenospermia were recruited in this study. The present study was approved by the Institutional Ethics Committees of West China Second Hospital. The study conforms with the Helsinki Declaration of 1975 (as revised in 2008) concerning Human and Animal Rights. All of the participants in this study signed the informed consent.

Quantitative polymerase chain reaction

Briefly, total RNA was extracted from tissue and cell samples using TransZol Up Plus RNA Kit (TransGen Biotech, ER501-01). Subsequently, the total RNA was reverse transcribed into double-stranded cDNA using SuperScript™ IV Reverse Transcriptase (Thermo Fisher, 18090010). Then the qPCR was performed in a total reaction volume of 10 µL containing 5 µL SYBR Green Mix (KCQS00, Sigma Aldrich), 3.6 µL DEPC-H2O, 0.2 µL forward primer, 0.2 µL reverse primer, and 1 µL cDNA (Total amount: 10 ng-100 ng). A two-step method was used: denaturation temperature of 95 °C for 30 s, annealing and extension of 60 °C for 30 s. The details were previously described [36]. The primers are shown in Tables S2 and S3.

Hematoxylin–eosin (HE) staining

The fixed testis tissues were embedded in paraffin and then cut into 5 µm ultrathin sections. The sections were then dewaxed in dimethylbenzene and dyed in hematoxylin for 50 s. After that, the sections were placed under flowing water for 5–10 min. Subsequently, the sections were immersed into 1% HCL 2–4 times and washed under flowing water as before. Next, the sections were immersed in eosin for 10 min and then dehydrated in gradient ethanol of 70, 80, 90, and 100%. Lastly, the stained sections were dried in a bake oven and then sealed with neutral balsam for subsequent observations.

Immunofluorescence staining

For staining of sperm and spermatogenic cells at different stages, the samples were first fixed in 4% paraformaldehyde and then homogeneously smeared onto the slides. Subsequently, the slides were punched in 0.5% Triton X-100 (Beyotime, P0096-100 m) for 10 min and then blocked in 5% BSA for 30 min at room temperature. After washing with phosphate buffered solution (PBS) twice, the slides were incubated overnight with primary antibodies (shown in Table S4) at 4 °C and then incubated with corresponding secondary antibodies at room temperature for 60 min. Subsequently, the slides were sealed with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, P0131) and observed under a laser confocal microscope (Olympus, FV1000).

For staining of testicular sections, the prepared 5 µm paraffin sections were dewaxed in dimethylbenzene and then immersed in sequential gradient ethanol (100, 100, 95, 85, 75, 50%) and distilled water. After the antigen repair in citrate sodium, the sections were then blocked in goat serum (Thermo Fisher Scientific, R37624) for 60 min at room temperature. Subsequently, the sections were successively incubated with primary antibodies overnight at 4 °C and secondary antibodies for 60 min at room temperature. After that, the sections were sealed with 5 µl DAPI and observed under a laser confocal microscope (Olympus, FV1000). The primary and secondary antibodies used in our research are shown in Table S4.

Isolation of single spermatogenic cell from mouse testis

The mice were executed and the fresh testes were isolated and placed in PBS. Subsequently, the albuginea was cut and removed. The testis tissues were then transferred to a 1.5 ml centrifuge tube containing 0.5 ml PBS and were completely sheared. Next, the tissue debris suspension passed through a 40 µm filter screen to obtain the single spermatogenic cells. The cell suspension was then centrifuged to obtain the precipitation of spermatogenic cells and the supernatant was discarded. The single spermatogenic cells were then fixed in 4% paraformaldehyde for subsequent experiments.

Flow cytometry of JC1, mitotracker, mitosox, and DHE staining

All of the detections were performed according to the operator’s instructions of JC1 (Beyotime, C2006), Mitotracker (Beyotime, C1049), Mitosox (Thermo Fisher, M36008), and DHE (Keygen, KGAF09) assay kits. Briefly, the living sperm or cells were stained with a working solution according to the corresponding concentration at 37 °C. After being washed twice with PBS, the samples were resuspended in a flow tube and then detected on a flow cytometer according to the corresponding excitation and emission wavelength.

Scanning electron microscopy and transmission electron microscopy

For SEM, fresh sperm isolated from cauda epididymis of mice were washed three times using PBS and then fixed on coverslips with 4% Paraformaldehyde for 2 h. Subsequently, the coverslips carrying spermatozoa were washed with PBS and then dehydrated with gradient ethanol of 50, 60, 70, 80, 95, and 100%. Then, the sperm were dried with Quorum K850 Critical Point Dryer (East Sussex, UK) and coated with gold particles using an ion sputter coater (Rotary Pumped Quorum Technologies, Q150RS). The spermatozoa were finally observed under SEM (Hitachi, S-3400N).

For TEM, testis tissues were isolated from mice and then fixed with 2.5% glutaraldehyde for more than 24 h. Subsequently, the samples were further fixed with 1% osmic acid for 120 min. After washing with PBS twice, the samples were dehydrated with gradient acetone of 50, 70, 90, and 100% at 4 °C and then embedded in embedding agents containing 6% butylene phthalate, 1% phenol, 44% dodecyl succinic anhydride, and 56% epoxy resin. Next, ultrathin sections (70–90 nm) were obtained and double-stained with lead citrate and uranyl acetate. Finally, the images were taken under transmission electron microscopy (Philips, TECNAI G2 F20) for further analysis.

Western blot

The details for western blot assay were previously described [36]. Briefly, collected cells and tissue samples were lysed on ice in RIPA lysis buffer for 60 min (spermatozoa lysed overnight at 4 °C) (Beyotime, P0013B), which contained the Protease Inhibitor Cocktail (Biomake, B14012). Subsequently, protein concentrations of each sample were determined by the BCA method (Solarbio, PC0020). Next, the same amounts of total proteins from 30 to 60 µg were loaded into each sampling hole and were separated via electrophoresis by 7.5–12.5% SDS-PAGE (Shanghai Epizyme Biomedical Technology Co., Ltd, PG112, PG113) and then transferred to polyvinylidene difluoride membranes (Millipore, ISEQ00010, IPVH00010). Subsequently, the membranes were successively incubated with primary antibodies overnight at 4 °C and secondary antibodies for 60 min at room temperature. Finally, the protein bands were visualized with ECL HRP substrate (Millipore, WBKLS0500). The GAPDH is used as the loading control to ensure that the total protein contents are consistent in each sampling well. The primary and secondary antibodies used in our research are shown in Table S4.

Co-Immunocoprecipitation

The detailed methods were previously described. In short, the extracted total proteins from the mouse testes were incubated with 5–10 µl target antibodies on the rotary shaker overnight at 4 °C. Subsequently, 50 µl of Protein A/G magnetic beads (Thermo Fisher, 88804) were added into each sample and incubated for 60 min at room temperature. After washing twice and resuspension in PBS, the proteins were eluted from the beads by denaturation in 1xSDS (Biosharp, BC502A) at 95 °C for 15 min. The obtained proteins were used for further western blot analysis.

Cell culture

HEK-293 T and HeLa cells were obtained from American type culture collection (ATCC) and cultured with DMEM (GIBCO, 11965092) containing 10% fetal bovine serum (Sigma, F8318) and 0.1% penicillin/streptomycin in an incubator with 5% CO2 at 37 °C. According to the experimental scheme, the overexpression (Abm, 382720940395) and knockdown (Bio-Atom Biotechnology, EV1420) plasmids of QRICH2 were transfected into the cells. After 48–72 h, the cells were collected to verify the overexpression or knockdown efficiency and conduct subsequent analysis. Furthermore, cells were cultured in the medium with absent Gln and collected for further assay after 72 h.

Determination of the concentrations of Gln and Glu

The concentrations of Gln and Glu were determined by colorimetry according to the users’ instructions (Suzhou Grace Biotechnology Co., Ltd, G0429W, G0427W). Gln/Glu were first freed by the lysis of the mouse testis tissues or spermatozoa. Subsequently, the kit ingredients and the free Gln/Glu were mixed in the tubes to produce a color reaction and then the absorbance was recorded by the Microplate Reader according to the maximum absorption wavelength (MOLECULAR DEVICES. CmaxPlus). Finally, the Gln/Glu concentrations were calculated based on the standard curve.

Amino acid metabonomic analysis

Metabonomic analysis was performed according to the standard experimental process.

The free amino acid mixtures were isolated from the testis tissues and then analyzed by ultra high performance liquid chromatography (Shimadzu, Nexera X2 LC-30AD) and mass spectrometry (AB SCIEX, 5500QTRAP). Subsequently, the chromatographic peak area and retention time were extracted by MultiQuant software and the 20 amino acid standards were used to correct the retention time and identify the metabolites. Two groups including WT and Qrich2 KO were analyzed and equal mixed samples of different samples were prepared to ensure the quality control and stability of the analysis in this study.

Microtubule polymerization experiments

The microtubule polymerization experiments were conducted as previously described [14]. The purified α-TUBULIN (HTS03) and GTP stock (BST06-1) were obtained from Cytoskeleton. The reactions were performed in 384 well plates in a volume of 50 µl. The General Tubulin Buffer (containing 4 mg/ml α-TUBULIN) based reaction systems of blank control, positive control, and sample control group were added into the plate and 1 mM GTP (final concentration) was added to prime the reactions. Immediately, the plate was placed into the preheated Microplate Reader to record the results. The absorption wavelength was set at 340 nm and the absorbance was recorded every 15 s. Paclitaxel (AbMole Bio science, 33069-62-4) at a 10 µM concentration was used as a positive control for promoting microtubule polymerization.

Single cell metabolic analysis

The analysis was performed according to the operator's manual. The cells were prepared according to the experimental requirements. Subsequently, the cells were digested and inoculated on the confocal dish at a density of 10%. On the second day, the Mitotracker staining was conducted as previously described and then the confocal dish was placed in the Single Cell Analyzer TM (JiangSu RayMe Biotechnology Co., Ltd, SCA500). Next, the focal length and the position of the light probe were adjusted to focus on a single cell to detect the fluorescence intensity.

Separation of sperm from Qrich2 heterozygous mice using the upstream method

First, the cauda epididymis was separated from the heterozygous mice and then placed in the 1.5 ml EP tube containing 1 ml TYH (Easy check, M2030). An incision was produced to enable the swimming out and capacitation of the spermatozoa. Next, the tube was vertically placed in the incubator at 37 °C for 60 min. Subsequently, the 300-µl upper and lower sperm suspension were sucked out using a pipettor.

The sperm suspensions were then centrifuged at 2000 rpm for 5 min to obtain the sperm for further analysis.

The enriched ontology clusters analysis

The enriched ontology clusters analysis was performed using the online tool Metascape (http://metascape.org). Submitting the gene list and selecting the appropriate species to be aligned for analysis.

The construction of the phylogenetic tree of QRICH2

Twenty-one homologous genes of QRICH2 were obtained from 12 species, including Acipenser ruthenus, Rhacophorus reinwardtii, Xenopus laevis, Anolis carolinensis, Chrysemys picta, Thamnophis elegans, Anas platyrhynchos, Taeniopygia guttata, Melopsittacus undulate, Ailuropoda melanoleuca, Homo sapiens, and Mus musculus (As shown in Table S5). Because the C16orf96 protein has a similar domain of DUF4795 with QRICH2, we set C16orf96 as an out group of the phylogenetic tree. We performed multiple alignments using Multiple Sequence Comparison by Log-Expectation, conducted the alignment curation using Cleaning aligned sequences, and finally constructed the phylogenetic tree using a method of Maximum likelihood-based inference of phylogenetic trees with Smart Model Selection. Moreover, we used the NCBI database to label the conserved domains of the obtained QRICH2 and C16orf96 proteins and merged the phylogenetic tree with the domain.

Duolink proximity ligation assay

The PLA assay was conducted using the Duolink In Situ reagents (DUO92008, Sigma) according to the manual instructions. First, sperm cells were fixed with 4% PFA on the slides and permeabilized with 0.3% Triton X-100 in PBS for 20 min. Then the slides were blocked with 10% goat serum for 1 h at room temperature and incubated with rabbit anti-QRICH2 and mouse anti-α-TUBULIN primary antibodies. Later, the oligonucleotide-labeled anti-rabbit and anti-mouse PLA probes were used to label the primary antibodies. If the protein–protein interactions in situ are within a 40 nm distance, a PLA red fluorescent signal is generated. Subsequently, the slides were washed with PBS and mounted using anti-fade mounting media with DAPI. The images were captured using a laser confocal microscope (Olympus FV3000).

Protein structure prediction and protein–protein docking

Three-dimensional structural models of QRICH2 and TUBULIN alpha-3 chain were downloaded from the AlphaFold Protein Structure Database [12, 30] (https://alphafold.com/). Flexible docking between QRICH2 and TUBULIN alpha-3 chain was performed on the HADDOCK webserver (https://bianca.science.uu.nl/haddock2.4/submit/1) [29]. To define the ambiguous interaction restraints (AIRs) in docking, the active and passive residues of QRICH2 and TUBULIN alpha-3 chain were predicted by the CASTp webserver (http://sts.bioe.uic.edu/castp/calculation.html) [26]. The first complex model with the lowest HADDOCK score was chosen to be the final model. And the protein–protein interaction was analyzed by LIGPLOT [33]. The structure diagram was made by PyMOL software. Furthermore, conserved motifs were predicted by MEME (Multiple EM for Motif Elicitation) motif discovery tool (http://meme-suite.org/tools/meme) [2].

Detection of cellular ATP levels

ATP levels in sperm, testis, and cell lysates were measured using the luminometer method according to the manufacturer's instructions (S0027, Beyotime Biotechnology). For normalization, total proteins were extracted from samples before ATP assay.

The detection of cellular α-ketoglutarate levels

The concentration of α-Ketoglutarate in samples was detected by high-performance liquid chromatography method following the manufacturer’s instructions for α-Ketoglutarate HPLC Assay kit (BC4704, Solarbio Science & Technology Co., Ltd., Beijing, China).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.4.0 software and SPSS 17.0 software. All of the data were presented as the means ± SEM. P < 0.05 was considered to be statistically significant. Statistical significance between the two groups was calculated using an unpaired, parametric, two-sided Student’s t-test.