Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility.

Varying 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 calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, 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 CATSPERζ 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.

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 (TM) domain-containing protein, CATSPERθ is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore-forming subunit CATSPER4. CATSPERθ 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 CATSPERθ 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 TM subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might act 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 CATSPERθ in sperm motility and male fertility.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

CatSper Calcium Channels: 20 Years On.

The flagellar-specific Ca2+ channel CatSper is the predominant Ca2+ entry site in mammalian sperm. CatSper-mediated Ca2+ signaling affects nearly every event that regulates sperm to acquire fertilizing capability. In this review, we summarize some of the main findings from 20 years of CatSper research and highlight recent progress and prospects.

Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca2+ signaling.

Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+ ) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+ -signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux.

Hyperglycemia adversely affects critical physiological events related to rat sperm capacitation.

Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (EmR, DiSC3(5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na+ ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while EmR was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Sperm plasma membrane ion transporters and male fertility potential: A perspective under the prism of cryopreservation.

Intracellular calcium homeostasis plays a crucial role in spermatozoa by regulating physiological functions associated with sperm quality and male fertility potential. Intracellular calcium fine balance in the sperm cytoplasm is strictly dependent on sperm surface channels including the CatSper channel. CatSpers' role is to ensure the influx of extracellular calcium, while intracellular pH alkalinization serves as a stimulus for the activation of several channels, including CatSper. Overall, the generation of intracellular calcium spikes through CatSper is essential for fertilization-related processes, such as sperm hyperactivation, acrosome reaction, egg chemotaxis, and zona pellucida penetration. Multiple lines of evidence suggest that disruption in the close interaction among ions, pH, and CatSper could impair male fertility potential. In contemporary times, the growing reliance on Medically Assisted Reproduction procedures underscores the impact of cryopreservation on gametes. In fact, a large body of literature raises concerns about the cryo-damages provoked by the freeze-thawing processes, that can affect the plasma membrane integrity, thus the structure of pivotal ion channels, and the fine regulation of both intracellular calcium and pH. This review aims to provide an overview of the importance of the CatSper channel in sperm quality and further fertilization potential. Additionally, it addresses the emerging issue of cryopreservation's impact on the functionality of this sperm channel.

Environmental pollutants and male infertility: Effects on CatSper.

Infertility is a growing health concern among many couples worldwide. Men account for half of infertility cases. CatSper, a sperm-specific Ca2+ channel, is expressed on the cell membrane of mammalian sperm. CatSper plays an important role in male fertility because it facilitates the entry of Ca2+ necessary for the rapid change in sperm motility, thereby allowing it to navigate the hurdles of the female reproductive tract and successfully locate the egg. Many pollutants present in the environment have been shown to affect the functions of CatSper and sperm, which is a matter of capital importance to understanding and solving male infertility issues. Environmental pollutants can act as partial agonists or inhibitors of CatSper or exhibit a synergistic effect. In this article, we briefly describe the structure, functions, and regulatory mechanisms of CatSper, and discuss the body of literature covering the effects of environmental pollutants on CatSper.

The Chemosensing Role of CatSper in Mammalian Sperm: An Updated Review.

After sperm enter the female reproductive tract, the physicochemical and biochemical microenvironment undergoes significant changes. In particular, the large changes in various ions encountered by sperm may alter the physiology of sperm, ultimately compromising capacitation and fertilization. Thus, the rapid response to environmental variations is vital for sperm functions. For example, Calcium, the most crucial ion for sperm functions, enters into sperm via Ca2+ permeable ion channels. The cation channel of sperm (CatSper) is a sperm-specific, pH-sensitive, and Ca2+-permeable ion channel. It is responsible for the predominant Ca2+ entry in mammalian sperm and is involved in nearly every event of sperm to acquire fertilizing capability. In addition, CatSper also serves as a pivotal polymodal chemosensor in mammalian sperm by responding to multiple chemical cues. Physiological chemicals (such as progesterone, prostaglandins, ß-defensins, and odorants) provoke Ca2+ entry into sperm by activating CatSper and thus triggering sperm functions. Additionally, synthetic and natural chemicals (such as medicines, endocrine disrupting chemicals, drugs of abuse, and antioxidants) affect sperm functions by regulating CatSper-dependent Ca2+ signaling. Therefore, understanding the interactions between CatSper and extracellular ligands sheds light on the mechanisms underlying male infertility and offers innovative diagnostic and treatment approaches. This underscores the importance of CatSper as a crucial regulatory target in male reproduction, linking sperm function with the extracellular environment. In conclusion, this review comprehensively summarizes the relevant studies describing the environmental factors that affect CatSper in humans and rodents.

SKF96365 modulates activity of CatSper channels in human sperm.

Exposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+ concentration ([Ca2+]i) transient followed by repetitive [Ca2+]i activity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P = 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]i transient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]i increase and reversibly arrested [Ca2+]i oscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]i transient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P = 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.

CatSper and its CaM-like Ca2+ sensor EFCAB9 are necessary for the path chirality of sperm.

Successful fertilization depends on sperm motility adaptation. Ejaculated and activated sperm beat symmetrically in high frequency, move linearly, and swim with clockwise chirality. After capacitation, sperm beat asymmetrically with lower amplitude and a high lateral head excursion. This motility change called hyperactivation requires CatSper activation and an increase in intracellular Ca2+ . However, whether CatSper-mediated Ca2+ influx participates in controlling the swim path chirality is unknown. In this study, we show that the clockwise path chirality is preserved in mouse sperm regardless of capacitation state but is lost in the sperm either lacking the entire CatSper channel or its Ca2+ sensor EFCAB9. Pharmacological inhibition of CatSper with either mibefradil or NNC 55-0396 leads to the same loss in swim path chirality. Exposure of sperm to the recombinant N-terminal part of the zona pellucida protein 2 randomizes chirality in capacitated cells, but not in non-capacitated ones. We conclude that Ca2+ sensitive regulation of CatSper activity orchestrates clockwise swim path chirality of sperm and any substantial change, such as the physiological stimulus of zona pellucida glycoproteins, results in a loss of chirality.

Sperm calcium flux and membrane potential hyperpolarization observed in the Mexican big-eared bat Corynorhinus mexicanus.

Mammalian sperm capacitation involves biochemical and physiological changes, such as an increase in intracellular calcium ion concentration ([Ca2+]i), hyperpolarization of the plasma membrane potential and sperm hyperactivation, among others. These changes provide sperm with the ability to fertilize. In the bat Corynorhinus mexicanus, there is an asynchrony between spermatogenesis and sperm storage in the male with the receptivity of the female. For instance, in C. mexicanus, spermatogenesis occurs before the reproductive season. During the reproductive period, sperm are stored in the epididymis for a few months and the testis undergoes a regression, indicating low or almost null sperm production. Therefore, it is unclear whether the elements necessary for sperm fertilization success undergo maturation or preparation during epididymis storage. Here, we characterized pH-sensitive motility hyperactivation and Ca2+ influx in sperm, regulated by alkalinization and progesterone. In addition, by electrophysiological recordings, we registered currents that were stimulated by alkalinization and inhibited by RU1968 (a CatSper-specific inhibitor), strongly suggesting that these currents were evoked via CatSper, a sperm Ca2+-specific channel indispensable for mammalian fertilization. We also found hyperpolarization of the membrane potential, such as in other mammalian species, which increased according to the month of capture, reaching the biggest hyperpolarization during the mating season. In conclusion, our results suggest that C. mexicanus sperm have functional CatSper and undergo a capacitation-like process such as in other mammals, particularly Ca2+ influx and membrane potential hyperpolarization.

Reconstruction of the birth of a male sex chromosome present in Atlantic herring.

The mechanisms underlying sex determination are astonishingly plastic. Particularly the triggers for the molecular machinery, which recalls either the male or female developmental program, are highly variable and have evolved independently and repeatedly. Fish show a huge variety of sex determination systems, including both genetic and environmental triggers. The advent of sex chromosomes is assumed to stabilize genetic sex determination. However, because sex chromosomes are notoriously cluttered with repetitive DNA and pseudogenes, the study of their evolution is hampered. Here we reconstruct the birth of a Y chromosome present in the Atlantic herring. The region is tiny (230 kb) and contains only three intact genes. The candidate male-determining gene BMPR1BBY encodes a truncated form of a BMP1B receptor, which originated by gene duplication and translocation and underwent rapid protein evolution. BMPR1BBY phosphorylates SMADs in the absence of ligand and thus has the potential to induce testis formation. The Y region also contains two genes encoding subunits of the sperm-specific Ca2+ channel CatSper required for male fertility. The herring Y chromosome conforms with a characteristic feature of many sex chromosomes, namely, suppressed recombination between a sex-determining factor and genes that are beneficial for the given sex. However, the herring Y differs from other sex chromosomes in that suppression of recombination is restricted to an ∼500-kb region harboring the male-specific and sex-associated regions. As a consequence, any degeneration on the herring Y chromosome is restricted to those genes located in the small region affected by suppressed recombination.

Metagenomics approach for Polymyxa betae genome assembly enables comparative analysis towards deciphering the intracellular parasitic lifestyle of the plasmodiophorids.

Genomic knowledge of the tree of life is biased to specific groups of organisms. For example, only six full genomes are currently available in the rhizaria clade. Here, we have applied metagenomic techniques enabling the assembly of the genome of Polymyxa betae (Rhizaria, Plasmodiophorida) RES F41 isolate from unpurified zoospore holobiont and comparison with the A26-41 isolate. Furthermore, the first P. betae mitochondrial genome was assembled. The two P. betae nuclear genomes were highly similar, each with just ~10.2 k predicted protein coding genes, ~3% of which were unique to each isolate. Extending genomic comparisons revealed a greater overlap with Spongospora subterranea than with Plasmodiophora brassicae, including orthologs of the mammalian cation channel sperm-associated proteins, raising some intriguing questions about zoospore physiology. This work validates our metagenomics pipeline for eukaryote genome assembly from unpurified samples and enriches plasmodiophorid genomics; providing the first full annotation of the P. betae genome.

Heat stress disrupts spermatogenesis via modulation of sperm-specific calcium channels in rats.

Heat is a detrimental environmental stressor that disrupts spermatogenesis and results in male infertility. Previous investigations have shown that heat stress reduces the motility, number, and fertilization ability of living spermatozoa. Sperm hyperactivation, capacitation, acrosomal reaction, and chemotaxis towards the ova are regulated by the cation channel of sperm (CatSper). This sperm-specific ion channel triggers the influx of calcium ions into sperm cells. The aim of this study in rats was to investigate whether heat treatment affected the expression levels of CatSper-1 and -2, together with the sperm parameters, testicular histology and weight. The rats were exposed to heat stress for 6 days and the cauda epididymis and testis were collected 1, 14, and 35 days after heat treatment to measure sperm parameters, gene and protein expression, testicular weight, and histology. Interestingly, we found that heat treatment caused a notable downregulation of CatSper-1 and -2 expression at all three time points. In addition, there were significant reductions in sperm motility and number and an increase in the percentage of abnormal sperm at 1 and 14 days, with cessation of sperm production at 35 days. Furthermore, expression of the steroidogenesis regulator, 3 beta-hydroxysteroid dehydrogenase (3ß-HSD) was upregulated in the 1-, 14- and 35-day samples. Heat treatment also upregulated the expression of the apoptosis regulator, BCL2-associated X protein (BAX), decreased testicular weight, and altered testicular histology. Therefore, our data showed for the first time that heat stress downregulated CatSper-1 and -2 in the rat testis, and that this may be a mechanism involved in heat stress-induced impairment of spermatogenesis.

The Cation/Calcium Channel of Sperm (CatSper): A Common Role Played Despite Inter-Species Variation?

The main cation/calcium channel of spermatozoa (CatSper), first identified in 2001, has been thoroughly studied to elucidate its composition and function, while its distribution among species and sperm sources is yet incomplete. CatSper is composed of several subunits that build a pore-forming calcium channel, mainly activated in vivo in ejaculated sperm cells by intracellular alkalinization and progesterone, as suggested by the in vitro examinations. The CatSper channel relevance is dual: to maintain sperm homeostasis (alongside the plethora of membrane channels present) as well as being involved in pre-fertilization events, such as sperm capacitation, hyperactivation of sperm motility and the acrosome reaction, with remarkable species differences. Interestingly, the observed variations in CatSper localization in the plasma membrane seem to depend on the source of the sperm cells explored (i.e., epididymal or ejaculated, immature or mature, processed or not), the method used for examination and, particularly, on the specificity of the antibodies employed. In addition, despite multiple findings showing the relevance of CatSper in fertilization, few studies have studied CatSper as a biomarker to fine-tune diagnosis of sub-fertility in livestock or even consider its potential to control fertilization in plague animals, a more ethically defensible strategy than implicating CatSper to pharmacologically modify male-related fertility control in humans, pets or wild animals. This review describes inter- and intra-species differences in the localization, structure and function of the CatSper channel, calling for caution when considering its potential manipulation for fertility control or improvement.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17ß-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Human sperm cells can form paracetamol metabolite AM404 that directly interferes with sperm calcium signalling and function through a CatSper-dependent mechanism.

STUDY QUESTION: Do paracetamol (N-acetyl-para-aminophenol (APAP) or acetaminophen) and/or its metabolites affect human sperm Ca2+-signalling and function? SUMMARY ANSWER: While APAP itself does not interact with Ca2+-signalling in human sperm, its metabolite N-arachidonoyl phenolamine (AM404), produced via fatty acid amide hydrolase (FAAH), interferes with human sperm Ca2+-signalling and function through a suggested CatSper channel-dependent action. WHAT IS KNOWN ALREADY: Studies have shown that adult men with high urinary levels of over-the-counter mild analgesic APAP have impaired sperm motility and increased time-to-pregnancy. STUDY DESIGN, SIZE, DURATION: This study consists of (i) an in vivo human pharmaceutical APAP exposure experiment to understand to what degree APAP reaches the sperm cells in the seminal fluid; (ii) in vitro calcium imaging and functional experiments in freshly donated human sperm cells to investigate CatSper channel-dependent activation by APAP and its metabolites; and (iii) experiments to understand the in situ capabilities of human sperm cells to form APAP metabolite AM404. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three healthy young males participated in the in vivo human exposure experiment after prior consent. Human semen samples were provided by healthy young volunteer donors after prior consent on the day of the in vitro experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Pharmaceutical APAP exposure reaches the seminal plasma in high micromolar concentrations and accumulates in the seminal plasma between 3 and 5 days of exposure (P-value 0.023). APAP and its primary metabolite 4-aminophenol (4AP) do not interact with human sperm Ca2+-signalling. Instead, the APAP metabolite AM404 produced via FAAH interferes with human sperm Ca2+-signalling through a CatSper-dependent action. Also, AM404 significantly increases sperm cell penetration into viscous mucous (P-value of 0.003). FAAH is functionally expressed in human sperm cells in the neck/midpiece region, as evidenced by immunohistochemical staining and the ability of human sperm cells to hydrolyse the fluorogenic FAAH substrate arachidonyl 7-amino, 4-methyl coumarin amide in an FAAH-dependent manner. Importantly, human sperm cells have the capacity to form AM404 in situ after exposure to 4AP (P-value 0.0402 compared to vehicle-treated sperm cells). LIMITATIONS, REASONS FOR CAUTION: The experiments were conducted largely in vitro. Future studies are needed to test whether APAP can disrupt human sperm function in vivo through the action of AM404. WIDER IMPLICATIONS OF THE FINDINGS: We hypothesize that these observations could, at least in part, be responsible for the negative association between male urinary APAP concentrations, sperm motility and time-to-pregnancy. STUDY FUNDING/COMPETING INTEREST(S): D.M.K. is funded by the Lundbeck Foundation, grant number R324-2019-1881, and the Svend Andersen Foundation. A.R. is funded by a BRIDGE-Translational Excellence Programme grant funded by the Novo Nordisk Foundation, grant agreement number: NNF18SA0034956. All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.