- Open Access
Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations
Reproductive Biology and Endocrinology volume 20, Article number: 5 (2022)
Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated.
We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy.
We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition.
SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.
Infertility is a significant global health issue affecting approximately 10–15% of couples worldwide, with the male factor being a component in at least 50% of couples. Studies have shown that many genetic and epigenetic factors play a role in male infertility, but many cases occur due to unknown reasons .
Many factors have been found to influence sperm motility and morphology, and genetic defects in the sperm result in the development of asthenoteratozoospermia . Potassium channels are important factors in the structure and function of the sperm, and their defects can cause dysfunction in the physiology and function of sperm. As a potassium transporter, potassium channel subfamily U1 (KCNU1, also known as SLO3) has a high level of expression in the testis, and its deletion has been reported to lead to defects in sperm motility and fertility in mouse [3, 4]. In particular, SLO3, first cloned in mouse spermatocytes in 1998, belongs to the high conductance Slo K+ channels family and is a sperm-specific potassium channel whose physiological function has been confirmed by whole-cell patch clamping techniques . Similar to SLO1, the calcium and voltage-gated potassium channel, SLO3 consists of a voltage-dependent pore formed by four identical α-subunits and a large cytoplasmic C-terminal, which has been proposed to be the ligand-binding sensor . Both the pH and voltage dependence as well as the properties of the single channel of SLO3 have been well defined . Despite the low sequence conservation of SLO3 among different mammalian species, it displays a high testis-specific expression pattern, located in the main part of the flagellum of sperm in mice [8, 9]. In addition, SLO3 has been reported to be sensitive to both pH and the voltage level and to play a role in sperm capacitation, sperm motility, and acrosome reaction. Deletion of the gene coding this ion channel might result in defective sperm activation and fertility in mice [3, 4, 10, 11].
So far, studies on the function of SLO3 have primarily focused on the mouse orthologue, mSLO3. Electrophysiological analysis and genetic deletion studies established that mSLO3 is a voltage- and pH-gated channel that mediates most K+ current in murine sperm. The function of mSLO3 has been shown to be essential for male fertility as Slo3 knockout (KO) mice were found to be infertile, with sperm cells exhibiting a series of functional defects. In humans, it is unknown whether SLO3 is functionally expressed in the sperm and serves a similar key role for the normal morphology and motility of the sperm and fertilization. Some reports have studied the properties of the hSLO3 channel by using the heterologous expression of hSLO3 channels in Xenopus oocytes [12, 13]. However, the exact functional properties of hSLO3 have not been characterized in human sperm. In this study, we analysed genetic data obtained by whole-exome sequencing (WES) from a large cohort of 105 patients with asthenoteratozoospermia, and identified a single patient carrying a homozygous missense mutation in SLO3. The adult male harbouring the homozygous SLO3 variant was sterile, and characterized by abnormal sperm morphology and reduced sperm motility. However, intra-cytoplasmic sperm injection (ICSI) treatment using the mutant spermatozoa led to successful fertilization. Therefore, this work provided the first evidence that absence of SLO3 causes male infertility due to abnormalities in sperm morphology, the mitochondrial sheath, acrosome reaction, and membrane potential of sperm in human.
Materials and methods
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.
SLO3 is involved in human male infertility with asthenoteratozoospermia
We performed WES on a cohort of 105 individuals with severe asthenoteratozoospermia. After applying stringent filters criteria according to our previously described protocol, we identified a single individual (A-132) carrying a homozygous mutation in SLO3 (GenBank: NM_001031836.3, c.1237A > T: p.Ile413Phe). The SLO3 mutation was confirmed by Sanger sequencing (Fig. 1A). The subject A-132 from a consanguineous family, and presented with severely reduced sperm motility and a comparatively low sperm count (12.7 × 106/mL) (Table 2, Fig. 1A). We noticed that the allele frequency of the SLO3 variant was < 1% in any database, including 1000 Genomes Project and gnomAD (v2.1.1 with 141,456 samples). Moreover, the PolyPhen-2, SIFT, and MutationTaster tools predicted it to be deleterious (Table 1).
The SLO3 (also known as KCNU1) gene is located on human chromosome 8 and contains 27 exons encoding a predicted 1149-amino acid potassium channel protein, specifically expressed in human spermatozoa. The SLO family channels are composed of 4 transmembrane voltage sensor domains (VSDs) surrounding a central pore gate domain (PGD), and a large cytosolic domain known as the gating ring. The gating ring is assembled from the regulators of K+ conductance (RCK1 and RCK2) domains of each of the 4 subunits, forming a ring with a central opening. The RCK domain determines the closed vs open conformational state of the gating ring and transduces the effects of any factor interacting with the gating ring to the PGD. Interestingly, the residue in the RCK domain of the SLO3 protein affected by the SLO3 missense variant p. Ile413Phe is highly conserved in SLO3 orthologues (Fig. 1B). Further analysis of protein structural modelling using online bioinformatic tools revealed the severe effects of this amino acid-substituting mutation on the structure of the SLO3 protein, indicating that this mutation introduces an amino acid with different properties, which may disturb this domain and abolish its function (Fig. 1C).
Morphological abnormalities in sperm heads and mid-piece flagella of the man harbouring the homozygous SLO3 variant
We examined sperm morphological and ultrastructural changes in sperm of SLO3 mutant subject. Compared with the sperm from a fertile control individual, the spermatozoa from the individual harbouring the SLO3 variant displayed a high rate of head malformations, in particular, an abnormal acrosomal region, indicating a defect in acrosome formation. In addition, we observed that sperm tails displayed swollen midpieces or shorter principal pieces of highly irregular width, indicating poorly assembled mitochondrial sheaths (Fig. 2A and Table 2). When observed by SEM, we found that spermatozoa of unaffected controls exhibited a smooth, regularly contoured, oval-shaped head and a long flagellum with a clearly defined midpiece, whereas most spermatozoa from the individual with the SLO3 variant exhibited severe morphological defects (Fig. 2B), such as swollen midpieces (II and V), coil-shaped flagella (III), small acrosome (IV), absence of acrosome in combination with defective mid-piece (VI and VII), and insufficient chromatin condensation (VIII).
To better understand the nature of these head and flagella defects observed in the spermatozoa with the SLO3 mutation, we analysed their ultrastructure. We accordingly found that their acrosomes were either absent or morphologically defective (Fig. 2C). We also noticed that most acrosomes were quite thin, showing highly diminished contents with barely recognizable inner and outer acrosomal membranes. Furthermore, a higher incidence of insufficient chromatin condensation was also observed in the spermatozoa of the affected individual compared with those of controls. The longitudinal sections of the flagellar mid-piece of spermatozoa in the individual harbouring the homozygous SLO3 variant showed a seriously disorganized or short mitochondrial sheath compared with those of control (Fig. 2C). However, we noticed that in cross-sections, the arrangements of ODF, DMT, and CP were not significantly different (Fig. 2D). Indeed, many mutant spermatozoa had a coil-shaped flagella that might have been associated with deficient osmoregulation and volume control, a condition expected to result from the mutation-induced ablation of the ion channel.
To further characterize the molecular defects induced by the SLO3 mutation in human sperm, we examined the expression of SLO3 mRNA and protein in sperm samples from control and individuals harbouring the SLO3 variant. As shown in Fig. 3A, the SLO3 mRNA was significantly (P < 0.001) reduced in spermatozoa from the man harbouring the SLO3 variant, as well as the protein expression level (Fig. 3B and C), indicating that the observed sperm defects were probably induced by the deficiency in SLO3.
SLO3 deficiency affected the functions of acrosome and mitochondria
The ultrastructure analysis of spermatozoa from the individual harbouring the SLO3 variant indicated that SLO3 might play an essential role in acrosome formation and mitochondrial sheath assembly. To investigate the effect of the SLO3 variant on the spermatozoa acrosome state, we evaluated the percentage of spontaneous acrosome-reacted cells using PSA-FITC binding. We found a significant increase (Student’s t-test; P < 0.001) in the rate of acrosome reaction in spermatozoa of the man harbouring the SLO3 variant compared with that of controls (Fig. 4A and B). In addition, we also observed that most spermatozoa from controls presented a highly stained PNA in acrosomal region, whereas the signal was absent in most spermatozoa from the SLO3 mutant individual. Moreover, the localization patterns of PLCζ1 were located in the acrosomal region in most spermatozoa from control individuals (Fig. 4C). However, spermatozoa from the subject harbouring the SLO3 variant showed a predominant base of the neck and middle-piece of the tail localization pattern, indicating that both the formation of the acrosome and the localization pattern of PLCζ1 were influenced by the SLO3 deficiency.
Furthermore, we investigated the effect of SLO3 deficiency on mitochondrial function. As shown in Fig. 5A, compared with control, the mitochondrial membrane potential was drastically decreased in spermatozoa from the man harbouring the SLO3 variant. Subsequently, we examined the expression of HSP60 in the spermatozoa of the affected individual with that of healthy controls. We found that the expression of HSP60 was significantly reduced in the mitochondrial sheath components (Fig. 5B and C), in consistency with the sperm motility and the TEM results for spermatozoa of the affected individual. These results demonstrated that SLO3 plays a key role in spermiogenesis by upholding the integrity of acrosomal and mitochondrial sheath components.
The hyperpolarization of sperm from individual harbouring SLO3 variant was decreased during capacitation
In mouse sperm, deletion of the Slo3 gene abolished alkalization-activated K+ current . Thus, we wondered whether the SLO3 homozygous mutant would alter the membrane potential of sperm during capacitation. Consistently, we found that the hyperpolarization of sperm from individual harbouring SLO3 variant was decreased significantly during capacitation (Fig. 6A). To understand the molecular mechanism of the observed lack of membrane potential after capacitation in the SLO3 mutant sperm, we examined the expression of the SLO3 auxiliary subunit LRRC52 in sperm cells from control and the SLO3-mutant individual Compared with normal controls (Fig. 6B and C), we noticed that the expression of LRRC52 was significantly decreased in sperm cells from the SLO3-mutant individual. Furthermore, we also investigated the sperm-specific change in CatSper expression in the spermatozoa of the SLO3 mutated individual. We specifically observed that immunostaining of CatSper1 in the spermatozoa from the individual harbouring the SLO3 variant was comparable to that observed in control sperm (Fig. 6B and D), suggesting that the Ca2+-activated K+ currents were controlled by LRRC52 and not CatSper1.
SLO3 deficiency-related male infertility could be rescued by intracytoplasmic sperm injection treatment
It has been previously reported that Slo3−/− male mice are infertile, but Slo3−/− sperm exhibits some fertility when utilized in vitro fertilization assays. In addition, ICSI has been suggested as an effective way to circumvent the physical limitations experienced by asthenoteratozoospermia sperm in our hospital. The individual with the SLO3 variant was 29 years old at treatment, while his partner was 34 years old. As a couple, they underwent 1 ovarian stimulation cycle, with the number of metaphase II oocytes retrieved and injected being 15. We observed that the rates of the fertilization, cleavage, 8-cell and blastocyst formation ware comparable to those in OAT controls (Table 3). Consecutively, 9 blastocysts were frozen. Six months later, 2 blastocysts were transferred and a single foetal heart beat was detected via ultrasound, with the pregnancy being currently ongoing. Therefore, our study indicated that male infertility caused by a homozygous SLO3 variant could be rescued by ICSI treatment.
To the best of our knowledge, we demonstrated for the first time that the presence of a homozygous mutation of SLO3 could induce severe morphological defects in the head and midpiece of human spermatozoa, together with a deficiency in the acrosome reaction and impaired progressive motility in sperm. The SLO3 protein localizes in the flagellum and is especially concentrated at the flagellar midpiece of spermatozoa, possibly playing an important role in mitochondrial sheath assembly. In addition, when observed by light microscopy and SEM, most spermatozoa from the individual harbouring the SLO3 variant exhibited angular/bent tails compared with those of healthy controls, consistent with the observations in Slo3−/− mouse sperm . However, in cross-sections, the arrangements of ODF, DMT, and CP were not significantly different, which might have been associated with a deficient osmoregulation and volume control in sperm, a condition probably resulting from the mutation-induced ablation of a major ion channel .
The eukaryotic SLO1 and SLO3 proteins exhibit a high amino acid sequence identity (42% identity), and, in fact, sequence analysis has suggested that SLO3 evolved from a duplication of the SLO1 gene [6, 22]. However, SLO1 and SLO3 channels share a unique property among the extended family of voltage-gated K+ channels in that their opening requires the synergistic action of membrane depolarization and specific intracellular cues . The SLO1 gene is expressed in excitable cells, such as neurons or muscles . Whereas SLO3, which is exclusively expressed in mammalian sperm, is a voltage- and pH-gated channel that mediates most K+ current in murine sperm essential to male fertility and is activated by an increase in intracellular pH [3, 23]. In humans, the exact functional properties of hSLO3 have not been characterized. By heterologously expressing human SLO3 in Xenopus oocytes, MacKinnon et al. found that the pH sensitivities of hSLO3 and mSLO3 were comparable . Whereas Strünker et al. observed that the membrane potential in human sperm was weakly activated by pH and more strongly by Ca2+, a finding that differed from that observed for the mSlo3 channel . In our study, the SLO3 mutation impaired the membrane potential of mutant sperm after capacitation. In addition, the expression of the SLO3 auxiliary subunit LRRC52 was significantly reduced in sperm from the SLO3 mutant individual compared with that from control, in accordance with the finding that LRRC52 was completely absent from Slo3−/− sperm . This result indicated that the expression of LRRC52 is critically dependent on the presence of SLO3 [24,25,26]. Moreover, the expression of CatSper1 was similar in both control and effected sperm, indicating that Ca2+ channels were not affected by the mutation in SLO3.
It has been reported that Slo3−/− male mice were unable to fertilize wild-type females, despite the presence of fertilization plugs . However, partial IVF success was achieved with oocytes harvested from super ovulating wild-type females incubated with capacitated sperm from Slo3−/− male mice . Besides, ICSI has become an effective method to help infertile couples, particular for infertile men with asthenoteratozoospermia, achieve a successful pregnancy. In this study, we noticed that the fertilization, cleavage, 8-cell and blastocyst formation rates were similar when performing ICSI with sperm from individual harbouring SLO3 variant and control sperm. We froze 9 blastocysts and 6 months later, transferred 2 blastocysts, resulting in the detection of a single foetal heartbeat via ultrasound; the pregnancy is ongoing. Therefore, our study indicated that male infertility caused by a homozygous SLO3 variant could be rescued by ICSI treatment, which is meaningful for the genetic counselling of patients with SLO3-associated asthenoteratozoospermia.
In conclusion, our findings on the homozygous mutation of SLO3 might further broaden our knowledge on genetic pathologies associated with male infertility due to severe asthenoteratozoospermia. Our findings might also have important clinical implications for the genetic and reproductive counselling of affected families. Deletion of SLO3 would seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. However, there were certain limitations in the present study. Further investigations identifying the functional role of SLO3 in acrosome formation, mitochondrial sheath assembly are required, and such investigations might involve the screening of a large number of patients with severe asthenoteratozoospermia.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Haematoxylin and eosin
Scanning electron microscopy
Transmission electron microscopy
Intra-cytoplasmic sperm injection
Quantitative real-time PCR
Miller D, Vukina J. Recent advances in clinical diagnosis and treatment of male factor infertility. Postgrad Med. 2020;132:28–34.
Saberiyan M, Mirfakhraie R, Gholami D, Dehdehi L, Teimori H. Investigating the regulatory function of the ANO1-AS2 on the ANO1 gene in infertile men with asthenozoospermia and terato-asthenozoospermia. Exp Mol Pathol. 2020;117:104528.
Santi CM, Martinez-Lopez P, de la Vega-Beltran JL, Butler A, Alisio A, Darszon A, et al. The SLO3 sperm-specific potassium channel plays a vital role in male fertility. FEBS Lett. 2010;584:1041–6.
Zeng XH, Yang C, Kim ST, Lingle CJ, Xia XM. Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa. Proc Natl Acad Sci U S A. 2011;108:5879–84.
Schreiber M, Wei A, Yuan A, Gaut J, Saito M, Salkoff L. Slo3, a novel pH-sensitive K+ channel from mammalian spermatocytes. J Biol Chem. 1998;273:3509–16.
Leonetti MD, Yuan P, Hsiung Y, Mackinnon R. Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel. Proc Natl Acad Sci U S A. 2012;109:19274–9.
Zeng XH, Navarro B, Xia XM, Clapham DE, Lingle CJ. Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa. J Gen Physiol. 2013;142:305–13.
Geng Y, Ferreira JJ, Dzikunu V, Butler A, Lybaert P, Yuan P, et al. A genetic variant of the sperm-specific SLO3 K(+) channel has altered pH and ca (2+) sensitivities. J Biol Chem. 2017;292:8978–87.
Santi CM, Butler A, Kuhn J, Wei A, Salkoff L. Bovine and mouse SLO3 K+ channels: evolutionary divergence points to an RCK1 region of critical function. J Biol Chem. 2009;284:21589–98.
De La Vega-Beltran JL, Sanchez-Cardenas C, Krapf D, Hernandez-Gonzalez EO, Wertheimer E, Trevino CL, et al. Mouse sperm membrane potential hyperpolarization is necessary and sufficient to prepare sperm for the acrosome reaction. J Biol Chem. 2012;287:44384–93.
Demarco IA, Espinosa F, Edwards J, Sosnik J, De La Vega-Beltran JL, Hockensmith JW, et al. Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation. J Biol Chem. 2003;278:7001–9.
Brenker C, Zhou Y, Muller A, Echeverry FA, Trotschel C, Poetsch A, et al. The Ca2+−activated K+ current of human sperm is mediated by Slo3. Elife. 2014;3:e01438.
Lopez-Gonzalez I, Torres-Rodriguez P, Sanchez-Carranza O, Solis-Lopez A, Santi CM, Darszon A, et al. Membrane hyperpolarization during human sperm capacitation. Mol Hum Reprod. 2014;20:619–29.
Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HW, Behre HM, et al. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16:231–45.
Bjorndahl L, Barratt CL, Mortimer D, Jouannet P. ‘How to count sperm properly’: checklist for acceptability of studies based on human semen analysis. Hum Reprod. 2016;31:227–32.
He X, Liu C, Yang X, Lv M, Ni X, Li Q, et al. Bi-allelic loss-of-function variants in CFAP58 cause flagellar Axoneme and mitochondrial sheath defects and Asthenoteratozoospermia in humans and mice. Am J Hum Genet. 2020;107:514–26.
Auger J, Jouannet P, Eustache F. Another look at human sperm morphology. Hum Reprod. 2016;31:10–23.
Tang S, Wang X, Li W, Yang X, Li Z, Liu W, et al. Biallelic mutations in CFAP43 and CFAP44 cause male infertility with multiple morphological abnormalities of the sperm flagella. Am J Hum Genet. 2017;100:854–64.
Liu C, He X, Liu W, Yang S, Wang L, Li W, et al. Bi-allelic mutations in TTC29 cause male subfertility with Asthenoteratospermia in humans and mice. Am J Hum Genet. 2019;105:1168–81.
He X, Li W, Wu H, Lv M, Liu W, Liu C, et al. Novel homozygous CFAP69 mutations in humans and mice cause severe asthenoteratospermia with multiple morphological abnormalities of the sperm flagella. J Med Genet. 2019;56:96–103.
Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11:307–11.
Vicens A, Vinuesa P, Arenas M, Trevino CL. Analyzing the functional divergence of Slo1 and Slo3 channel subfamilies. Mol Phylogenet Evol. 2019;133:33–41.
Zhang X, Zeng X, Lingle CJ. Slo3 K+ channels: voltage and pH dependence of macroscopic currents. J Gen Physiol. 2006;128:317–36.
Yang C, Zeng XH, Zhou Y, Xia XM, Lingle CJ. LRRC52 (leucine-rich-repeat-containing protein 52), a testis-specific auxiliary subunit of the alkalization-activated Slo3 channel. Proc Natl Acad Sci U S A. 2011;108:19419–24.
Kawai T, Miyata H, Nakanishi H, Sakata S, Morioka S, Sasaki J, et al. Polarized PtdIns (4,5) P2 distribution mediated by a voltage-sensing phosphatase (VSP) regulates sperm motility. Proc Natl Acad Sci U S A. 2019;116:26020–8.
Zeng XH, Yang C, Xia XM, Liu M, Lingle CJ. SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility. Proc Natl Acad Sci U S A. 2015;112:2599–604.
We would like to thank the families for participating and supporting this study. We also thank the Center of Cryo-Electron Microscopy at Zhejiang University.
This work was supported by the National Natural Science Foundation of China (82071705, 81971441 and 31521003), the open project of NHC Key Laboratory of Male Reproduction and Genetics (KF201907), the University Outstanding Young Talents Support Program (gxyq2021174), Major Science and Technology Project of Anhui Province (202003a07020012), and Scientific Research (TP202002) from Anhui Medical University, the University Synergy Innovation Program of Anhui Province (GXXT-2021-071).
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This study was approved and consented by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University.
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The authors declare no competing interests.
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Lv, M., Liu, C., Ma, C. et al. Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations. Reprod Biol Endocrinol 20, 5 (2022). https://doi.org/10.1186/s12958-021-00880-4
- Acrosome hypoplasia
- Mitochondrial sheath malformation