Biology subjects

Chung, J.-J.

Publications and source records attributed to Chung, J.-J..

14 recordsLinked to original sources

bioRxiv2026

Soluble ZP2 N-terminal fragments activate CatSper-dependent Ca2+ entry and regulate motility and acrosomal exocytosis in mammalian sperm

Sperm motility and function are central to mammalian fertilization and are tightly regulated by intracellular calcium (CaCa2+) signaling. This signaling is primarily orchestrated by the CatSper Ca2+; channel complex located in the flagella of spermatozoa. However, the natural ligands that activate CatSper remain largely unknown in many species, despite the conservation of CatSper in mammals. Here, we present a signaling role for soluble N-terminal ZP2 fragments in regulating CatSper activity and sperm physiology in mice and humans. ZP2 has been implicated in mediating sperm binding and recognition at the oocyte surface interface; however, new evidence is starting to unveil the molecular mechanisms and function of ZP2 during fertilization transition. Here, we show that the during fertilization, cleaved ZP2 N-terminal fragment triggers a rapid and robust increase in intracellular CaCa2+ levels in sperm. This increase depends strictly on CatSper function, as demonstrated through pharmacological analysis and CatSper1 knockout mice. This calcium influx is sufficient to induce acrosomal exocytosis in a subset of human and mouse sperm. AlphaFold-based structural modeling suggests a potentially conserved extracellular interaction site between the soluble N-terminal ZP2 fragments and the CatSper complex. In human sperm, ZP2 treatment significantly modulates motility parameters, including flagellar movement and velocity, while inducing a CatSper-dependent increase in intracellular CaCa2+ similar in magnitude to that evoked by progesterone. Species-matched ZP2 stimulation elicits the stronger calcium response, underscoring evolutionary adaptations in ligand-channel protein pairs. Taken together, our findings reveal a conserved signaling pathway from ZP2 to CatSper that integrates oocyte-derived signals into the regulation of sperm motility and acrosomal exocytosis. This pathway provides new mechanistic insights into fertilization and highlights potential targets.

cell biology↗

bioRxiv2026

CATSPERβ--δ Interaction Governs Hierarchical CatSper Holo-complex Assembly and is Essential for Male Fertility

The sperm-specific CatSper channel is macromolecular Ca2+ channel complex essential for hyperactivated motility and male fertility. The pore-forming channel (CATSPER1-4) associates with a large extracellular domain-containing canopy (CATSPER{beta}-{varepsilon}), a cytosolic Ca{superscript 2} sensing subcomplex (CATSPER{zeta}-EFCAB9-ARMH2), the putative transporter SLCO6C1, and CATSPER{theta}-, arranging into higher-order zigzag rows in the flagellar membrane. However, the molecular mechanism governing holo-complex assembly during spermatogenesis remains largely undefined. Here we demonstrate that the CATSPER{beta}-{delta} interaction represents an essential early step in CatSper biogenesis. CRISPR/Cas9 targeting of Catsperb exon 4 generated a frameshift knockout (Catsperb-/-) and an in-frame deletion mutant (Catsperb{Delta}/{Delta}) that specifically disrupts the {beta}-{delta} interaction interface. Disrupting this interface reduced CATSPER{delta} among canopy subunits, impairing canopy assembly and destabilizing the core pore-forming channel. Consequently, mature spermatozoa completely lacked the entire CatSper complex, phenocopying the knockout. A transgenic line expressing extracellular-domain-truncated CATSPER{delta} lacking the {beta}-binding region likewise phenocopied the Catsperb mutant, confirming the necessity of an intact {beta}-{delta} interface. AlphaFold-Multimer modeling and alanine-substitution mutagenesis identified key hydrogen-bonding residues at this interface that mediate canopy subunit association. Consistent with complex loss, whole-sperm patch-clamp recordings revealed complete absence of CatSper conductance in mutant spermatozoa. Both mutant lines exhibited defective sperm hyperactivation and male infertility despite normal spermatogenesis and baseline motility. Together, these findings establish that canopy formation driven by CATSPER{beta}-{delta} interaction precedes and is required for pore-forming channel assembly, defining a hierarchical assembly pathway for the CatSper holo-complex and highlighting a key structural node for male fertility and contraceptive development.

cell biology↗

bioRxiv2025

Coordinated and compartmentalized functions of CAII and CAIV establish a dual pH-regulatory mechanism essential for sperm maturation and capacitation

Carbonic anhydrase II (CAII) and IV (CAIV) orchestrate pH regulation essential for male fertility through distinct cellular compartments, with CAII functioning intracellularly and CAIV GPI-anchored extracellularly in epididymal and vas deferens epithelia as well as on spermatozoa. These enzymes control pH environments required for sperm maturation and capacitation by regulating bicarbonate availability, which triggers soluble adenylyl cyclase/cAMP signaling, elevates intracellular pH (pHi), and enables motility activation. However, the specific contributions of CAII and CAIV to luminal pH and sperm pHi regulation remain unclear. Here, we show that genetic ablation of either CAII or CAIV in mice impairs normal luminal acidification in the male reproductive tract and unexpectedly lowers the basal pHi of sperm. This lower basal pHi blunts subsequent intracellular alkalinization, thereby diminishing the activation of pH-sensitive CatSper Ca2+ channels essential for hyperactivated motility. Mechanistically, we demonstrate that this basal pHi reduction occurs independently of the CatSper channel itself, but it is phenocopied in K+channel Slo3-deficient sperm, suggesting a homeostatic link between flagellar pH and membrane potential. Super-resolution imaging reveals that CAII is located within the flagellum near the CatSper channel in the principal piece, while CAIV is distributed along the entire flagellar membrane. Together, our findings demonstrate that CAII and CAIV employ spatially coordinated mechanisms to regulate pH. By maintaining an acidic luminal environment for sperm maturation and priming the subsequent alkalinization of sperm pHi, these enzymes modulate downstream ion channel activity to govern sperm motility and fertility. One-sentence summarySpatially coordinated carbonic anhydrases II and IV govern sperm motility by establishing the dual-compartment pH environments necessary to activate downstream flagellar ion channels.

cell biology↗

bioRxiv2024

Extracellular domains of CATSPERε are essential for CatSper assembly during development and activity modulation in sperm capacitation

The flagellar-specific Ca2+ channel CatSper is a multiprotein complex that is critical for successful fertilization by controlling the sperm Ca2+ signaling in space and time. Large extracellular domains (ECDs) of four single-pass transmembrane subunits, CATSPER{beta}, {gamma}, {delta}, and {varepsilon}, form a unique canopy structure over the pore-forming channel. However, the molecular mechanisms of canopy assembly during development and its physiological function in mature sperm remain unknown. Here, using two genetic mouse models and the biochemical isolation of a bioactive CATSPER{varepsilon} fragment, we report that CATSPER{varepsilon} ECDs are essential for assembling the CatSper canopy, and thus the entire channel complex, and for modulating CatSper function for sperm hyperactivation and fertilization. CATSPER{varepsilon}-deficient males are sterile because their sperm fail to develop hyperactivated motility due to the absence of the entire channel. In transgenic mice overexpressing CATSPER{varepsilon} with truncated ECDs in testicular germ cells, truncated CATSPER{varepsilon} is unable to interact with native CatSper subunits and incorporate into the complex, thus failing to rescue the defective sperm hyperactivation and infertility of Catspere-null males. These findings provide insight into the underlying molecular and developmental mechanisms of CatSper complex assembly and how CatSper channels can be modulated in physiological settings and by therapeutic intervention.

cell biology↗

bioRxiv2023

DHCR24-mediated sterol homeostasis during spermatogenesis is required for sperm mitochondrial sheath formation and impacts male fertility over time

Desmosterol and cholesterol are essential lipid components of the sperm plasma membrane. Cholesterol efflux is required for capacitation, a process through which sperm acquire fertilizing ability. In this study, using a transgenic mouse model overexpressing 24-dehydrocholesterol reductase (DHCR24), an enzyme in the sterol biosynthesis pathway responsible for the conversion of desmosterol to cholesterol, we show that disruption of sterol homeostasis during spermatogenesis led to defective sperm morphology characterized by incomplete mitochondrial packing in the midpiece, reduced sperm count and motility, and a decline in male fertility with increasing paternal age, without changes in body fat composition. Sperm depleted of desmosterol exhibit inefficiency in the acrosome reaction, metabolic dysfunction, and an inability to fertilize the egg. These findings provide molecular insights into sterol homeostasis for sperm capacitation and its impact on male fertility.

cell biology↗

bioRxiv2023

A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane domain containing protein, CATSPER{theta}, is essential for the CatSper channel assembly during sperm tail formation. CATSPER{theta} facilitates the channel assembly by serving as a scaffold for a pore forming subunit CATSPER4. CATSPER{theta} is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPER{theta} are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper transmembrane subunits results in loss of CATSPER{theta} protein in the spermatid cells during spermatogenesis. CATSPER{theta} might acts as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPER{theta} in sperm motility and male fertility.

cell biology↗

bioRxiv2023

LRRC23 loss-of-function impairs radial spoke 3 head assembly and causes defective sperm motility underlying male infertility

Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23. A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males.

cell biology↗

bioRxiv2021

TXNRD3 supports male fertility via the redox control of spermatogenesis

Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

biochemistry↗

bioRxiv2021

A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and while its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo, using developing spermatogenic cells as a model. Remarkably, we find that AGO2 acts in the germ cell nucleus to positively regulate protein expression. We show that AGO2 dynamically binds both chromatin and nuclear mRNA transcripts of hundreds of genes required for sperm production, and germline conditional knockout (cKO) of Ago2 causes depletion of the corresponding proteins, along with defects in sperm number and morphology. Nuclear AGO2 partners with splicing, export, and chromatin factors to promote transcript export and protein expression. Together, our data reveal an unexpected role for nuclear AGO2 in enhancing expression of developmentally important genes.

developmental biology↗

bioRxiv2021

C2cd6-encoded CatSper{tau} Targets Sperm Calcium Channel to Ca2+ Signaling Domains in the Flagellar Membrane

In mammalian sperm cells, regulation of spatiotemporal Ca2+ signaling relies on the quadrilinear Ca2+ signaling nanodomains in the flagellar membrane. The sperm-specific, multi-subunit CatSper Ca2+ channel, which is crucial for sperm hyperactivated motility and male fertility, organizes the nanodomains. Here, we report CatSper{tau}, the C2cd6-encoded membrane-associating C2 domain protein, can independently migrate to the flagella and serve as a major targeting component of the CatSper channel complex. CatSper{tau} loss-of-function in mice demonstrates that it is essential for sperm hyperactivated motility and male fertility. CatSper{tau} targets the CatSper channel into the quadrilinear nanodomains in the flagella of developing spermatids, whereas it is dispensable for functional channel assembly. CatSper{tau} interacts with ciliary trafficking machinery in a C2-dependent manner. These findings provide insights into the CatSper channel trafficking to the Ca2+ signaling nanodomains and the shared molecular mechanisms of ciliary and flagellar membrane targeting. HighlightsO_LICatSper{tau} encoded by C2cd6 is a C2 membrane-associating domain containing protein C_LIO_LICatSper{tau} loss-of-function impairs sperm hyperactivation and male fertility C_LIO_LICatSper{tau} adopts ciliary trafficking machineries for flagellar targeting via C2 domain C_LIO_LICatSper{tau} targets the CatSper channel into nanodomains of developing sperm flagella C_LI

cell biology↗

bioRxiv2021

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of redox regulation in sperm development and maturation remain largely elusive. In this study, we report that TXNRD3, a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation, regulates redox homeostasis to support male reproduction. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility under conditions of TXNRD3 deficiency. We reveal that mitochondria develop more defined cristae during capacitation in wild type sperm. Absence of TXNRD3 alters redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

physiology↗

bioRxiv2021

3D structure and in situ arrangements of CatSper channel in the sperm flagellum

The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca2+ gateway into the sperm flagellum. However, CatSpers complex subunit composition has impeded its reconstitution in vitro and structural elucidation. Here, we applied cryo-electron tomography to visualize the macromolecular organization of the native CatSper channel complex in intact mammalian sperm, as well as identified three additional CatSper-associated proteins. The repeating CatSper units form long zigzag-rows in four nanodomains along the flagella. In both mouse and human sperm, each CatSper repeat consists of a tetrameric pore complex. Murine CatSper contains an additional outwardly directed wing-structure connected to the tetrameric channel. The majority of the extracellular domains form a canopy above each pore-forming channel that interconnects to a zigzag-shaped roof. The intracellular domains link two neighboring channel complexes to a diagonal array. The loss of this intracellular link in Efcab9-/- sperm distorts the longitudinally aligned zigzag pattern and compromises flagellar movement. This work offers unique insights into the mechanisms underlying the assembly and transport of the CatSper complex to generate the nanodomains and provides a long-sought structural basis for understanding CatSper function in the regulation of sperm motility.

biophysics↗

bioRxiv2020

3D in situ imaging of female reproductive tract reveals molecular signatures of fertilizing spermatozoa in mice

Out of millions of ejaculated sperm, only a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here, using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that all spermatozoa past the UTJ possess intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

cell biology↗

bioRxiv2018

EFCAB9 is a pH-Dependent Ca2+ Sensor that Regulates CatSper Channel Activity and Sperm Motility

SummaryVarying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calciumbinding domain-containing protein 9 (EFCAB9) as a dual function, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CAT SPER{zeta}, is essential for pH-dependent and Ca2+ sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPER{zeta} in a Ca2+ dependent manner and dissociates at elevated pH.These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.\n\nHighlightsO_LIEfcab9 encodes an evolutionarily conserved, sperm-specific EF-hand domain protein\nC_LIO_LIEfcab9-deficient mice have sperm motility defects and reduced male fertility\nC_LIO_LIEFCAB9 is a pH-tuned Ca2+ sensor for flagellar CatSper Ca2+ channel\nC_LIO_LIEFCAB9 is a dual function machine in gatekeeping and domain organization of CatSper\nC_LI

cell biology↗