In this study, we recruited a total of 105 Chinese men with asthenoteratozoospermia from the First Affiliated Hospital of Anhui Medical University. All 105 idiopathic infertile men were diagnosed with primary infertility for > 1 year. The individuals were recruited according to the guidelines of the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen . Individuals with obvious primary ciliary dyskinesia-related symptoms, such as bronchitis, sinusitis, otitis media, or pneumonia, as well as infertility caused by reproductive malformation, drugs, or exposure to gonadotoxic factors, were excluded. Individuals with abnormalities in their karyotype (46,XY) or Y chromosome microdeletions were also excluded. Peripheral whole blood samples were collected from all recruited participants for subsequent genetic analysis.
This study was approved by the ethics committee of the First Affiliated Hospital of Anhui Medical University. Informed consent was obtained from all participants and their family members, as well as from all fertile control male individuals.
Semen parameters and sperm morphological analysis
Semen samples from men with asthenoteratozoospermia and control subjects were collected via masturbation after 3–7 d of sexual abstinence and measured after liquefaction for 30 min at 37 °C in the source laboratories during the routine biological examination of individuals in accordance with the WHO guidelines (5th Edition) [14, 15]. Analyses of semen volume, sperm concentration, and motility were conducted during routine examination. Sperm morphology was analysed using haematoxylin and eosin (H&E) staining assay as previously described [16, 17], and 213 spermatozoa to evaluate the percentage of morphologically abnormal spermatozoa, such as small acrosome, swollen mid-piece, and coiled flagella. One spermatozoon was classified in only one morphological category according to its major abnormality.
Structural modeling for SLO3 and its mutants
The mutant Ile413Phe of SLO3 is located near the Pfam motifs, the effect on protein structure were modelled with homology models. 3D structural model of the SLO3 mutant was carried out using UCSF Chimera software, based on the 4HPF.pdb protein template.
Whole exome sequencing, bioinformatic analysis, and sanger sequencing
Genomic DNA was extracted from the peripheral blood of participants for WES analysis. Details on the methods used for library construction, WES, and data analysis were previously described . The SLO3 variant identified by WES was further validated by Sanger sequencing using the PCR primers presented in Table S1.
Scanning and transmission electron microscopy
For the electron microscopy assay, semen samples were prepared in accordance with a protocol previously described . Briefly, for scanning electron microscopy (SEM), samples were sequentially dehydrated using an ascending gradient of ethanol (Shengqiang Medical Technology, Jiangsu, China) and then dried with hexamethyldisilane (HMDS, Sigma-Aldrich, Castle Hill, NSW, Australia). Samples were then air-dried, added dropwise to specimen stubs, sputter coated, and examined using field emission SEM (Nova Nano 450, Thermo Fisher Scientific Inc., USA). For transmission electron microscopy (TEM), samples were fixed with 2.5% osmium tetroxide (Sigma-Aldrich, Castle Hill, NSW, Australia) and sequentially dehydrated using graded ethanol (50, 70, 90, and 100%) and 100% acetone (Sigma-Aldrich, Castle Hill, NSW, Australia). Samples were then infiltrated with acetone and SPI-Chem resin and embedded with Epon 812 (SPI#02659-AB, Structure Probe, USA). Subsequently, samples were sliced using an ultra-microtome (UC7, LEICA EM, Germany) and stained with uranyl acetate (#19481, Ted Pella, Inc., Redding, CA) and lead citrate (#19312, Ted Pella, Inc., Redding, CA). Cryoelectron microscopy (TecnaiG2 Spirit 120 kV, FEI, Netherlands) was used for image capturing.
Quantitative real-time PCR and western blotting
Total RNA from human spermatozoa was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA 92008 USA) and transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s protocol. The obtained cDNAs were used as templates for subsequent quantitative real-time PCR conducted using the Light Cycler 480 SYBR Green I Master (Roche, Switzerland, Germany). β-actin was used as an internal control. The assays were repeated thrice. Primers used were listed in Table S2.
Proteins from human sperm samples for immunoblotting were extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (89,842, Thermo Fisher Scientific Inc., USA) according to the manufacturer’s instructions. Briefly, washed sperm samples were suspended in 0.5 mL permeabilization buffer, vortexed, and incubated for 10 min at 4 °C with constant mixing. Permeabilized sperm samples were then centrifuged for 15 min at 16000 g and the supernatant containing cytosolic proteins was transferred to a new tube. The pellet was resuspended in 0.5 mL solubilization buffer and incubated for 30 min at 4 °C with mixing. Finally, resuspended samples were centrifuged at 16000 g for 15 min at 4 °C, and the supernatant containing solubilized membranes was collected and heated at 100 °C for 15 min. Lysates were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride (Pall Corporation, New York, NY, USA) membranes. Membranes were blocked in 5% non-fat milk diluted with TBST (TBS-0.1% Tween-20) for 1 h at 25 °C. Membranes were then immunoblotted using the following primary antibodies: rabbit polyclonal anti-LRRC52 (1:1000; PA5–107159, Invitrogen, Carlsbad, CA 92008 USA), rabbit polyclonal anti-CatSper1 (1:1000; DF9349, Affinity Biosciences, Beijing, China), rabbit polyclonal anti-Na+/K+-ATPaseα1 (1:2000; ABP51894, Abbkine, China), anti-HSP60 antibody (1:1000; ab13532, Abcam, Cambridge, UK), and HRP-conjugated β-actin (1:2000; HRP-60008, Proteintech, Rosemont, IL, USA), at 4 °C overnight. Signals were detected using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Beijing, China). Images were acquired using a CS analyser system (5200, Tanon, Shanghai, China). The Na+/K+-ATPase α1 or β-actin reference proteins were used as loading control.
Generation of polyclonal anti-SLO3 antibody
SLO3 polyclonal antibodies were generated by ABclonal Biotechnology in New Zealand rabbits using the 1120–1134 and 1156–1170 polypeptides of the human SLO3 protein (ENSP00000382770) as antigens. Briefly, the cDNA encoding these epitopes was cloned into a pET-28a expression vector, and the His-tagged fusion protein was expressed in Escherichia coli. The purified recombinant protein was used to generate polyclonal antisera in female New Zealand rabbits. Sequences of the peptides used were as follows: 1. SYQPRTNSLSFPKQ 2. KENERKTSDEVYDED.
Immunofluorescence staining of sperm samples was performed as previously described . Briefly, sperm samples were washed twice with phosphate buffer saline (PBS), fixed in 4% paraformaldehyde (Sigma-Aldrich, Castle Hill, NSW, Australia) at 4 °C overnight, and mounted on slides pre-treated with poly-L-lysine (Sigma-Aldrich, Castle Hill, NSW, Australia). Slides were incubated with primary antibodies (SLO3 (1:200, ABclonal Biotechnology, China), PLC-ζ1 (1:100, pab0367-P, covalab, USA), LRRC52 (1:500, PA5–107159, Invitrogen, Carlsbad, USA), CatSper1 (1:200; DF9349, Affinity Biosciences, Beijing, China), HSP60 (1:500; ab13532, Abcam, Cambridge, UK), acetylated alpha-tubulin (1:1000, mAb#5335, Cell Signaling Technology, Massachusetts, USA)) and PNA (1:500, RL-1072, VectorLabs, California, USA) overnight at 4 °C. After washing with PBS, slides were incubated with highly cross-adsorbed secondary Alexa Fluor 488 anti-mouse IgG (1:500, 34106ES60, Yeasen Biotechnology, USA) and Alexa Fluor 594 anti-rabbit IgG antibodies (1:500, 111–585-003, Jackson ImmunoResearch Inc., USA) for 1 h at 37 °C and subjected to Hoechst (1:1000, 62,249, Thermo Fisher Scientific Inc., USA) nuclear labelling for 2 h at 37 °C. Images were captured using an LSM 800 confocal microscope (CarlZeiss AG, Germany).
Detection of mitochondrial membrane potential by flow cytometry acquisition
Measurements of mitochondrial membrane potential (MMP) were performed using the lipophilic cationic dye 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanineiodide (JC-1) according to the manufacturer’s instructions (Invitrogen). Briefly, approximately 1 million sperm cells were washed with PBS, followed by incubation with 5 μM JC-1 working solution for 15 min at 37 °C. Samples were then washed and analysed by flow cytometry. For further analysis of all cytometric experiments, the debris was gated out based on light scattering measurements. For each analysis, at least 50,000 sperm cells were re-examined. All experiments were performed on a BD FACSVerse™ Flow Cytometer (BD Biosciences, USA). Flow cytometry acquisition for JC-1-stained sperm cells was performed through FL1 for green and FL2 for red fluorescence. At high MMP, JC-1 forms J-aggregates inside the mitochondria emitting red fluorescence, whereas in a low MMP state, it remains in the monomer form emitting green fluorescence.
Measurement of sperm membrane potential by flow cytometry
Sperm samples were washed twice with PBS. Pellets were then resuspended in Whitten’s HEPES-buffered media [(in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 20 HEPES, 5 glucose, 10 lactic acid, 1 Na-pyruvate] with or without 25 mM NaHCO3 and 5 g/L BSA and incubated with 1 μM potential-sensitive dye 3, 3′-dipropylthiocabocyanine iodine (DiSC3, Sigma-Aldrich, Castle Hill, NSW, Australia) for 8 min at 37 °C. After incubation, PI (Sigma-Aldrich, Castle Hill, NSW, Australia) was added and incubated for further 3 min at 37 °C. Before assaying the sperm using flow cytometry, 500 nM carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma-Aldrich, Castle Hill, NSW, Australia) was added in sperm suspensions and incubated at 37 °C for 2 min to dissipate the mitochondrial membrane potential. Analyses were conducted using a FACSVerse™ Flow Cytometer. Orange fluorescence from DiSC3-positive cells was detected at 600–700 nm and PI was detected at 500–560. Data were analyzed using FACS Diva and FlowJo software (Tree Star 9.3.3) as previously described . Cell debris, doublets and aggregates were excluded from analysis based on a dual parameter dot plot, in which pulse signal (signal high; FSC-H; y-axis) versus signal area (FSC-A; x-axis) was displayed.
The female partner of individual with SLO3 mutation had undergone a long protocol pituitary downregulation using GnRH agonist (Triptorelin, Diphereline 3.75 mg, Ipsen Pharma Biotech) and control ovarian hyperstimulation by recombinant FSH (Gonal-F; Serono) Oestradiol plasma levels and follicle growth were monitored every 2 days and human chorionic gonadotrophin (HCG, Livzon Pharmaceutical) was administered when three or more than follicles reached 18 mm in diameter. 36 h after HCG injection, 15 mature oocytes (MII) were retrieved for ICSI. Spermatozoa were prepared by discontinuous density gradient centrifugation and the resulting suspension was diluted in 10 μl drops of polyvinyl pyrolidine (PVP) covered with oil. Subsequently, metaphase II stage oocytes and motile sperm were selected for ICSI using a micromanipulator system (Olympus, Japan). Eighteen to 19 h later, the fertilized oocytes were assessed and cultured in cleavage medium (Cook, USA) in an incubator with an environment of 37 °C, 5% O2, 6% CO2, and 89% N2 until day 3 after fertilization. Then, the evaluated embryos were transferred to blastocyst medium (Cook, USA) and incubated to day 5 or day 6. According to the scoring system of Gardner and Schoolcraft , we obtained three day-5 blastocysts (4AB, 4BB and 3BB) and nine day-6 blastocysts (4BA, 4BB, 4 BC, 3BB*2 and 3CC*3), and nine blastocysts (3CC*3 poor blastocysts were discarded) were cryopreserved to prevent ovarian hyperstimulation syndrome (OHSS). After 6 months, two day-5 blastocysts were thawed and transferred in successive artificial cycles and a single foetal heart beat was detected via ultrasound after 28 days.
All data are representative of at least three independent experiments, and GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to perform the statistical analysis. Differences were analyzed by Student’s t-tests when comparing experimental groups, and P-values < 0.05 were considered significant.
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