Phylogenetic tree construction of Tex2
Data pertaining to TEX2/Tex2 sequences from various species were retrieved from the NCBI database (http://ncbi.nlm.nih.gov). The ClustalW tool, integrated within the MEGA-X software platform, facilitated the comparative analysis of multiple amino acid sequences. Subsequently, the TEX2 amino acid sequences underwent species analysis utilizing the neighbor-joining method, bolstered by 1000 bootstrap replicates for molecular evolutionary genetics inference. Concurrently, the UniProt database (https://www.uniprot.org/align/clustalo) was employed to evaluate the degree of similarity in amino acid sequences between mice and other species.
Ethical statement and knockout mice generation
All animal experimentation adhered to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. The protocols involving animal subjects received approval from the Experimental Animal Management and Ethics Committee of West China Second Hospital of Sichuan University (Project number: 20230719004).
Tex2 KO mice were generated by employing two sgRNAs targeting exon 3 and exon 4 of Tex2 in C57BL/6J genetic background mice using CRISPR/Cas9 knockout technology. Mouse genotyping was performed on DNA samples extracted from toe biopsies using Polymerase Chain Reaction (PCR). The amplification products yielded a length of 666 bp for WT genotypes and 450 bp for KO genotypes. The primer details were provided in Supplementary table 1.
Table 1. Semen analysis using CASA in the Tex2 KO mice.
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Adult male mice
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WT
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KO
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P*
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Semen parameters
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|
|
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Sperm concentration (10g/ml)
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65.19±7.08
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46.16±4.31
|
0.0834
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Motility (%)
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23.4±1.80
|
8.35±1.2
|
0.00230
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Progressive motility (%)
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21.5±1.10
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6.79±0.99
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0.00058
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Sperm locomotion parameters
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Curvilinear velocity (VCL) (um/s)
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20.67±2.90
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14.5±1.38
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0.128
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Straight-line velocity (VSL) (um/s)
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6.04±2.33
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2.15±0.55
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0.179
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Average path velocity (VAP) ((um/s)
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10.6±2.33
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4.77±0.69
|
0.073
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Amplitude of lateral head displacement (ALH)
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0.31±0.019
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0.25±0.02
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0.081
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Linearity (LIN)
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0.27±0.067
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0.147±0.029
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0.165
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Wobble (WOB, = VAP/VCL)
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0.503±0.038
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0.33±0.029
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0.052
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Straightness (STR, = VSL/VAP)
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0.53±0.091
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0.44±0.050
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0.418
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Beat-cross frequency (BCF) (Hz)
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2.48±0.16
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1.36±0.150
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0.069
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n = 3 biologically independent WT mice or KO mice.
RNA extraction and qPCR
Total RNA extraction was performed using Trizol reagent (9109, TaKaRa), followed by cDNA synthesis utilizing the PrimeScript RT kit (RR047A, TaKaRa). Subsequently, quantitative real-time polymerase chain reaction (qPCR) was conducted to amplify Actb (utilized as a control) and Tex2 employing SYBR Green Mix (KCQS00, Sigma-Aldrich), with the resultant products subjected to gel electrophoresis for analysis. Real-time fluorescence signals were captured utilizing an iCycler RT-PCR detection system from Bio-Rad Laboratories, and the obtained data were normalized using the 2-ΔΔCt method. Detailed primer sequences are provided in Supplementary table 1.
Western blotting
Testicular specimens were collected from male mice aged 8 weeks, representing both WT and Tex2 KO mice, and promptly placed on ice to maintain tissue integrity. Following harvest, the samples were lysed in RIPA buffer supplemented with a protease inhibitor, and subsequently centrifuged at 14,000 rpm for 15 minutes at 4°C. The concentration of testicular proteins was determined using the bicinchoninic acid (BCA) protein assay. Subsequently, the protein samples were mixed with SDS sample loading buffer, boiled for 10 minutes, and separated using 10% SDS-PAGE. The obtained proteins were transferred onto a PVDF membrane, and immunoblotting assays were conducted following established protocols[20]. The primary antibodies employed were rabbit anti-TEX2 (1:500, 12774-1-AP, Proteintech) and mouse anti-α-Tubulin (1:2000, ab4074, Abcam).
Fertility test
Sexually mature Tex2 KO mice, aged 8 weeks, were paired with WT C57BL/6J mice in a male-to-female ratio of 1:2, and these pairs were maintained for a minimum duration of 3 months. As a comparative control, WT males and WT females were co-housed under similar conditions. Continuous monitoring, systematic recording, and meticulous counting of litter sizes were diligently carried out for both Tex2 KO and WT female mice over the entire experimental period.
Papanicolaou staining
Mouse epididymal sperm underwent 2-3 washes with phosphate-buffered saline (PBS) before fixation and preservation in 4% formaldehyde-phosphate-buffered saline (FPA). For Papanicolaou staining of mouse spermatozoa, sperm from the epididymal tail were carefully collected and immersed in 95% ethanol for 15 minutes to facilitate fixation. Subsequently, rehydration was achieved through an ethanol gradient, and Papanicolaou staining (DA0191, Leagene Biotechnology) was executed following the manufacturer's instructions. Finally, the sperm morphology was scrutinized under a microscope.
Electron microscopy analyses
During scanning electron microscopy (SEM), spermatozoa were coated, fixed on slides, and subsequently rinsed twice with PBS. Subsequently, gradient dehydration was performed using various concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%). All dried samples were then affixed to aluminum mounts, platinum sputter-coated with an ion-spray instrument (Eiko E-1020, Hitachi), and ultimately examined using a field-emission SEM model, Hitachi S3400.
For transmission electron microscopy (TEM) assays, each semen sample underwent rinsing in sperm washing medium, post-fixation in 1% buffered osmium tetroxide, dehydration using a gradient acetone solution, and embedding in Epon 812. After localizing spermatozoa under a light microscope, ultrathin sections were double stained with lead citrate and uranyl acetate. Finally, the samples were analyzed using TEM (TECNAIG2F20, Philips) with an accelerating voltage of 80 kV. The methodology employed was previously described in the literature [21].
H&E staining
The testes and epididymis of male mice, as well as the ovaries of female mice, were removed following decapitation. These tissues were sampled and then fixed in a 4% PFA solution. Subsequently, the samples underwent dehydration using a series of ethanol concentrations (70%, 80%, 90%, 100%), were embedded in paraffin, and sectioned at a thickness of 5 μm. The resulting sections were then stained with hematoxylin and eosin (H&E) from Sigma-Aldrich, USA, and examined under a microscope. These procedures were concurrently conducted in both WT and Tex2 KO mice. The images obtained were analyzed using a LEICA DM2500 microscope from Germany and processed with CaseViewer software.
Chromosome spread
According to the available literature, the present experiments were conducted through the diffusion of spermatocytes, followed by immunofluorescence labeling, enabling the observation of chromosome morphology under the microscope [22]. Briefly, for this experiment, testes were harvested from WT and Tex2 KO mice at 8 weeks of age, respectively. The testicular leukomalacia was removed within half an hour, and the seminiferous tubules were placed in a hypotonic buffer (17 mM sodium citrate, 50 mM sucrose, 30 mM Tris of pH 8.2, 2.5 mM DL-dithiothreitol, 1 mM PMSF at pH 8.3, and 5 mM EDTA) for 30 minutes. Subsequently, the testicular cell suspension was dispersed onto a glass slide containing fixative (1% FPV, pH 9.2) and 0.15% Triton X-100. Anti-incubation antibodies, including SYCP3 (1:200, ab15093, Abcam) and γH2AX (1:200, ab124781, Abcam) primary antibodies, were then incubated at 37°C overnight. The secondary antibodies used for incubation were goat anti-mouse IgG, cross-adsorbed with Alexa Fluor 488(1:500, A21206, Thermo Fisher Scientific), and donkey anti-rabbit IgG (H+L), highly cross-adsorbed with Alexa Fluor 594 (1:500, A11005, Thermo Fisher Scientific). The incubation process lasted for one hour at 37°C, followed by 4,6-dibutyl-2-phenylindole (DAPI, F6057, Sigma-Aldrich) incubation for 30 minutes. Image acquisition was performed using a confocal microscope (Olympus FV3000, Japan).
Sperm motility analyses
In accordance with ethical guidelines, WT and Tex2 KO mice aged between 8 weeks were euthanized via cervical dislocation. Following euthanasia, the epididymal tail from one side of each mouse was promptly immersed in PBS maintained at 37°C for 5 minutes subsequent to neck dissection, facilitating the collection of spermatids. Thereafter, 10 μl of the collected tail was aspirated and transferred onto a sperm counting plate. Sperm motility was meticulously observed and assessed under an inverted microscope utilizing Computer-Assisted Sperm Analysis (CASA) technology. The results were recorded for further analysis, and the parameters tested included the percentage of motile sperm cells, mean path velocity (VAP), linear velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and beat crossover frequency (BCF).
Follicle counts
Paraffin-embedded ovaries underwent a meticulous procedure involving serial sectioning, paraffin clearance, and rehydration in preparation for standard H&E staining. Subsequently, follicle counts at various developmental stages in both WT and KO mice were conducted post-acquisition of H&E-stained images of the ovaries utilizing a microscope. The categorization of follicles into primordial, primary, secondary, and tertiary stages was based on established criteria [23]. The counting process specifically targeted follicles with discernible oocyte nuclei. The cumulative count of labeled follicles was then multiplied by 5 to derive the comprehensive count of follicles.
Immunofluorescence staining
For the immunofluorescence staining of mouse testis and ovary tissue, sections were prepared from previously preserved wax blocks containing embedded tissue. The paraffin sections underwent two consecutive treatments of deparaffinization in xylene for 10 minutes each, followed by hydration in graded alcohol concentrations (100%, 95%, 85%, 75%, and 50%). After antigen retrieval, the sections were blocked in a blocking solution for 30 minutes and then subjected to antibody incubation. For the immunofluorescence staining of mouse spermatozoa, a sample of spermatozoa preserved in 4% FPV was taken and coated onto slides to evenly distribute the spermatozoa. After the spermatozoa on the slides were dried and washed 2-3 times with PBS, 0.1% Triton X-100 was added to permeabilize them for 10 minutes. Subsequently, they were sealed with bovine serum albumin (BSA) in a dark box at room temperature for 1 hour, followed by antibody incubation. Mouse tissues and spermatozoa were stained by immunofluorescence using primary antibodies: rabbit anti-TEX2 (1:50, 12774-1-AP Proteintech) and mouse anti-α-Tubulin (1:200, ab4047, Abcam), which were incubated overnight at 4°C in a dark box. The next day, samples were incubated with Alexa Fluor 488 and Alexa Fluor 594 conjugated secondary antibodies for 1 hour at room temperature and stained with DAPI for 10 minutes. The above immunofluorescence staining preparations were conducted using Tex2 KO mice and WT mice over 2 months of age as controls. Images were captured using a confocal microscope (Olympus FV3000, Japan).
Statistical analysis
The results are expressed as the mean ± SEM. Statistical significance of variations among mean values for different genotypes was assessed through two-tailed unpaired Student’s t-tests conducted in GraphPad Prism 8. A P-value greater than 0.05 was deemed not statistically significant.