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Background: G‐protein‐coupled receptors are critical in many physiological and pathological processes in various organs. Serving as the control panel for sensing extracellular stimuli, G‐protein‐coupled receptors recognise various ligands, including light, temperature, odours, pheromones, hormones, neurotransmitters, chemokines, etc. Most recently, G‐protein‐coupled receptors residing in spermatozoa have been found to be indispensable for sperm function. Objective: Here, we have summarised cutting‐edge findings on the functional mechanisms of G‐protein‐coupled receptors that are known to be associated with sperm functions and the activation of their downstream effectors, providing new insights into the roles of G‐protein‐coupled receptors in sperm physiology. Results: Emerging studies hint that alterations in G‐protein‐coupled receptors could affect sperm function, implicating their role in fertility, but solid evidence needs to be continuing excavated with various means. Several members of the G‐protein‐coupled receptor superfamily, including olfactory receptors, opsins, orphan G‐protein‐coupled receptors, CXC chemokine receptor 4, CC chemokine receptor 5 and CC chemokine receptor 6 as well as their downstream effector β‐arrestins, etc., were suggested to be essential for sperm motility, capacitation, thermotaxis, chemotaxis, Ca2+ influx through CatSper channel and fertilisation capacity. Conclusion: The present review provides a comprehensive overview of studies describing G‐protein‐coupled receptors and their potential action in sperm function. We also present a critical discussion of these issues, and a possible framework for future investigations on the diverse ligands, biological functions and cell signalling of G‐protein‐coupled receptors in spermatozoa. Here, the G‐protein‐coupled receptors and their related G proteins that specifically were identified in spermatozoa were summarised, and provided references valuable for further illumination, despite the evidence that is not overwhelming in most cases. [ABSTRACT FROM AUTHOR]

ABSTRACTCa2+ is a key secondary messenger that determines sperm motility patterns. Mammalian sperm undergo capacitation, a process to acquire fertilizing ability, in the female reproductive tract. Capacitated sperm change their flagellar waveform to develop hyperactivated motility, which is crucial for successful sperm navigation to the eggs and fertilization. The sperm-specific channel, CATSPER, and an ATPase transporter, PMCA4, serve as major paths for Ca2+ influx and efflux, respectively, in sperm. The ionic paths coordinate Ca2+ homeostasis in the sperm, and their loss-of-function impairs sperm motility, to cause male infertility. In this review, we summarize the physiological significance of these two Ca2+ gates and suggest their potential applications in novel male contraceptives.

Background: The involvement of ATP and cAMP in sperm function has been extensively documented, but the understanding of the role of adenosine and adenosine receptors remains incomplete. This study aimed to examine the presence of adenosine A2A receptor (A2AR) and study the functional role of A2AR in human sperm. Methods: The presence and localization of A2AR in human sperm were examined by western blotting and immunofluorescence assays. The functional role of A2AR in sperm was assessed by incubating human sperm with an A2AR agonist (regadenoson) and an A2AR antagonist (SCH58261). The sperm level of A2AR was examined by western blotting in normozoospermic and asthenozoospermic men to evaluate the association of A2AR with sperm motility and in vitro fertilization (IVF) outcomes. Results: A2AR with a molecular weight of 43 kDa was detected in the tail of human sperm. SCH58261 decreased the motility, penetration ability, intracellular Ca2+ concentration, and CatSper current of human sperm. Although regadenoson did not affect these sperm parameters, it alleviated the adverse effects of SCH58261 on these parameters. In addition, the mean level of A2AR in sperm from asthenozoospermic men was lower than that in sperm from normozoospermic men. The sperm level of A2AR was positively correlated with progressive motility. Furthermore, the fertilization rate during IVF was lower in men with decreased sperm level of A2AR than in men with normal sperm level of A2AR. Conclusions: These results indicate that A2AR is important for human sperm motility and is associated with IVF outcome. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA), a typical endocrine disruptor, is known to have various adverse effects on the male reproductive system. However, the toxic effects and mechanisms of low-dose BPA have not yet been fully explored. In this study, male Kunming mice were orally administered low-dose BPA (0.03, 0.3 and 3 mg/kg/d) for ten consecutive weeks. Pathological sections of testicular tissue showed no significant morphological differences after BPA exposure. An analysis of the functional parameters of sperm revealed that exposure to low-dose BPA significantly decreased sperm motility, chemotaxis, and the acrosome reaction. An in vitro BPA exposure model combined with an omics data analysis showed that the olfactory receptor-related pathway was significantly enriched after BPA treatment. Subsequent experiments verified the reduced mRNA level of a novel olfactory receptor gene, Olfr25, in vivo and in vitro exposure models. Meanwhile, exposure to low-dose BPA reduced the intracellular calcium ion concentration and the mRNA levels of pore-forming subunits of the CatSper channel in sperm. Importantly, the knockdown of Olfr25 inhibited calcium ion levels and CatSper subunit expression in GC-2 cells. Olfr25 overexpression attenuated the BPA-induced downregulation of CatSper subunit expression in GC-2 cells. These findings indicate that Olfr25 might participate in low-dose BPA-induced sperm dysfunction by affecting the CatSper-Ca2+ signaling pathway. This study reveals a new mechanism underlying the effects of low-dose BPA on sperm function and provides a reference for assessing the safety of low-dose BPA exposure. [ABSTRACT FROM AUTHOR]

In female tract, mammalian sperm develop hyperactivated motility which is a key physiological event for sperm to fertilize eggs. This motility change is triggered by Ca2+ influx via the sperm-specific Ca2+ channel, CatSper. Although previous studies in human and mice largely contributed to understanding CatSper and Ca2+ signaling for sperm hyperactivation, the differences on their activation mechanisms are not well understood yet. There are several studies to examine expression and significance of the CatSper channel in non-human and non-mouse models, such as domestic animals. In this review, I summarize key knowledge for the CatSper channel from previous studies and propose future aspects for CatSper study using sperm from domestic animals.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specificCa2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSpermediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel. [ABSTRACT FROM AUTHOR]

From investigating molecular vibrations to observing galaxies, terahertz technology has found extensive applications in research and development over the past three decades. Terahertz time-domain spectroscopy and imaging have experienced significant growth and now dominate spectral observations ranging from 0.1 to 10 THz. However, the lack of standardised protocols for data processing, dissemination, and archiving poses challenges in collaborating and sharing terahertz data between research groups. To tackle these challenges, we present the dotTHz project, which introduces a standardised terahertz data format and the associated open-source tools for processing of dotTHz files. The dotTHz project aims to facilitate seamless data processing and analysis by providing a common framework. All software components are released under the MIT licence through GitHub repositories to encourage widespread adoption, modification, and collaboration. We invite the terahertz community to actively contribute to the dotTHz project, fostering the development of additional tools that encompass a greater breadth and depth of functionality. By working together, we can establish a comprehensive suite of resources that benefit the entire terahertz community. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA), a typical endocrine disruptor, is known to have various adverse effects on the male reproductive system. However, the toxic effects and mechanisms of low-dose BPA have not yet been fully explored. In this study, male Kunming mice were orally administered low-dose BPA (0.03, 0.3 and 3 mg/kg/d) for ten consecutive weeks. Pathological sections of testicular tissue showed no significant morphological differences after BPA exposure. An analysis of the functional parameters of sperm revealed that exposure to low-dose BPA significantly decreased sperm motility, chemotaxis, and the acrosome reaction. An in vitro BPA exposure model combined with an omics data analysis showed that the olfactory receptor-related pathway was significantly enriched after BPA treatment. Subsequent experiments verified the reduced mRNA level of a novel olfactory receptor gene, Olfr25, in vivo and in vitro exposure models. Meanwhile, exposure to low-dose BPA reduced the intracellular calcium ion concentration and the mRNA levels of pore-forming subunits of the CatSper channel in sperm. Importantly, the knockdown of Olfr25 inhibited calcium ion levels and CatSper subunit expression in GC-2 cells. Olfr25 overexpression attenuated the BPA-induced downregulation of CatSper subunit expression in GC-2 cells. These findings indicate that Olfr25 might participate in low-dose BPA-induced sperm dysfunction by affecting the CatSper-Ca2+ signaling pathway. This study reveals a new mechanism underlying the effects of low-dose BPA on sperm function and provides a reference for assessing the safety of low-dose BPA exposure.

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, β-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.

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specific Ca2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSper-mediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel.

[Display omitted] CatSper is a voltage dependent calcium ion channel present in the principal piece of sperm tail. It plays a crucial role in sperm hyperactivated motility and so in fertilization. Extracellular loops of mouse sperm CatSper were used to develop a vaccine to achieve protection from pregnancy. These loops were inserted at one of the three hypervariable regions of Human Papilloma Virus (HPV) capsid protein (L1). Recombinant vaccines were expressed in E.coli as inclusion body (IB), purified, refolded and assembled into virus-like particles (VLP) in vitro , and adsorbed on alum. Four vaccine candidates were tested in Balb/C mice. All the constructs proved immunogenic, one showed contraceptive efficacy. This recombinant contraceptive vaccine is a non-hormonal intervention and is expected to give long-acting protection from undesired pregnancies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration, and the sperm-specific Ca2+ channel CatSper has been shown to play an important role in the regulation of intracellular Ca2+. In particular, in mammals, CatSper is the only functional Ca2+ channel in the sperm, and mice deficient in the genes comprising the pore region of the Ca2+ channel are infertile due to the inhibition of sperm hyperactivation. CatSper is also thought to be involved in sea urchin chemotaxis. In contrast, in ascidian Ciona intestinalis, SAAF, a sperm attractant, interacts with Ca2+/ATPase, a Ca2+ pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is a functional Ca2+ channel in sperm.Results: We showed that CatSper is present in the sperm flagella of C. intestinalis as in mammalian species, although a small level of gene expression was found in other tissues. The spermatozoa of CatSper3 KO animals were significantly less motile, and some motile sperms did not show any chemotactic behavior. These results suggest that CatSper plays an important role in ascidians and mammals, and is involved in spermatogenesis and basic motility mechanisms.

Beta-defensin 20 (DEFB20) is widely expressed in the epididymis with gene features involved in epididymal sperm maturation. However, the action mechanism and function of DEFB20 in sperm maturation are still unclear. One of the important roles of beta-defensin is the ion channel activity. The cation channel sperm-associated protein (CatSper) alpha is an ion channel protein found on the sperm surface. This study aimed to investigate the interaction between DEFB20 and CatSper1-4 protein in relation to the sperm maturation process. Protein sequences were obtained from the National Center for Biotechnology Information (NCBI). Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method. Rosetta web server was used for the docking analysis. The results revealed a natural interaction between DEFB20 and CatSper1-4. The interaction occurred at the cation channel (close to the casein kinase II), ion transport protein, and kinase c phosphorylation of the CatSper1-4 active site. The DEFB20 region interacting with CatSper2-4 was the beta-defensin domain, while with CatSper1 was the non-beta-defensin domain. Based on the analysis, DEFB20 may interact with CatSper a subunits, particularly CatsSper1, to affect ion channel activity during sperm maturation. [ABSTRACT FROM AUTHOR]

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.

The flagellar-specific Ca21 channel CatSper is the predominant Ca21 entry site in mammalian sperm. CatSper-mediated Ca21 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. [ABSTRACT FROM AUTHOR]

The sperm-specific channel CatSper (cation channel of sperm) controls the intracellular Ca2+ concentration ([Ca2+]i) and plays an essential role in sperm function. It is mainly activated by the steroid progesterone (P4) but is also promiscuously activated by a wide range of synthetic and physiological compounds. These compounds include diverse steroids whose action on the channel is so far still controversial. To investigate the effect of these compounds on CatSper and sperm function, we developed a high-throughput screening (HTS) assay to measure changes in [Ca2+]i in human sperm and screened 1,280 approved and off-patent drugs including 90 steroids from the Prestwick chemical library. More than half of the steroids tested (53%) induced an increase in [Ca2+]i and reduced the P4-induced Ca2+ influx in human sperm in a dose-dependent manner. Ten of the most potent steroids (activating and P4-inhibiting) were selected for a detailed analysis of their action on CatSper and their ability to act on sperm acrosome reaction (AR) and penetration in viscous media. We found that these steroids show an inhibitory effect on P4 but not on prostaglandin E1-induced CatSper activation, suggesting that they compete for the same binding site as P4. Pregnenolone, dydrogesterone, epiandrosterone, nandrolone, and dehydroepiandrosterone acetate (DHEA) were found to activate CatSper at physiologically relevant concentrations within the nanomolar range. Like P4, most tested steroids did not significantly affect the AR while stanozolol and estropipate slightly increased sperm penetration into viscous medium. Furthermore, using a hybrid approach integrating pharmacophore analysis and statistical modelling, we were able to screen in silico for steroids that can activate the channel and define the physicochemical and structural properties required for a steroid to exhibit agonist activity against CatSper. Overall, our results indicate that not only physiological but also synthetic steroids can modulate the activity of CatSper with varying potency and if bound to CatSper prior to P4, could impair the timely CatSper activation necessary for proper fertilization to occur.

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.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different. [ABSTRACT FROM AUTHOR]

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 (Em R , DiSC 3 (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 Em R 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. [Display omitted] • Sperm from hyperglycemic rats respond to inducers of hyperactivation. • CatSper activity remains unchanged in sperm from hyperglycemic rats. • Hyperglycemia hyperpolarizes rat sperm. • Hyperglycemia depolarizes mitochondrial membrane potential in rat sperm. • Sperm from hyperglycemic rats exhibits low levels of ATP. [ABSTRACT FROM AUTHOR]

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.

Study questionDoes a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene?Summary answerPatient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE.What is known alreadyCatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex.Study design, size, durationThis was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ.Participants/materials, setting, methodsThe original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant.Main results and the role of chancePatient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious.Limitations, reasons for cautionThe nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated.Wider implications of the findingsPopulation genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives.Study funding/competing interest(s)This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

Ion channels control sperm navigation within the female reproductive tract and, thus, are critical for their ability to find and fertilize an egg. The flagellar calcium channel CatSper controls sperm hyperactivated motility and is dependent on an alkaline cytoplasmic pH. The latter is accomplished by either proton transporters or, in human sperm, via the voltage-gated proton channel Hv1. To provide concerted regulation, ion channels and their regulatory proteins must be compartmentalized. Here, we describe flagellar regulatory nanodomains comprised of Hv1, CatSper, and its regulatory protein ABHD2. Super-resolution microscopy revealed that Hv1 is distributed asymmetrically within bilateral longitudinal lines and that inhibition of this channel leads to a decrease in sperm rotation along the long axis. We suggest that specific distribution of flagellar nanodomains provides a structural basis for the selective activation of CatSper and subsequent flagellar rotation. The latter, together with hyperactivated motility, enhances the fertility of sperm.

Ion channels control the ability of human sperm to fertilize the egg by triggering hyperactivated motility, which is regulated by membrane potential, intracellular pH, and cytosolic calcium. Previous studies unraveled three essential ion channels that regulate these parameters: (1) the Ca2+ channel CatSper, (2) the K+ channel KSper, and (3) the H+ channel Hv1. However, the molecular identity of the sperm Na+ conductance that mediates initial membrane depolarization and, thus, triggers downstream signaling events is yet to be defined. Here, we functionally characterize DSper, the Depolarizing Channel of Sperm, as the temperature-activated channel TRPV4. It is functionally expressed at both mRNA and protein levels, while other temperature-sensitive TRPV channels are not functional in human sperm. DSper currents are activated by warm temperatures and mediate cation conductance, that shares a pharmacological profile reminiscent of TRPV4. Together, these results suggest that TRPV4 activation triggers initial membrane depolarization, facilitating both CatSper and Hv1 gating and, consequently, sperm hyperactivation.

To overcome egg protective vestments and ensure successful fertilization, mammalian spermatozoa switch symmetrical progressive motility to a powerful, whip-like flagellar motion, known as hyperactivation. The latter is triggered by a calcium influx through the sperm-specific, voltage-dependent, and alkalization-activated calcium channel of sperm - CatSper. The channel comprises nine subunits which together form a heteromeric complex. CatSper-deficient male mice and men with mutations in CatSper genes are infertile. This calcium channel is regulated by various endogenous compounds, such as steroids, prostaglandins, endocannabinoids, and intracellular pH. Being a sperm-specific ion channel that is not expressed anywhere else in the body, CatSper represents an ideal target for the development of female and even male contraceptives. In this review, we discuss the recent advances in studying CatSper functional properties and discuss future steps that are required to take in order to achieve a deep understanding of the molecular basis of CatSper function.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success. [ABSTRACT FROM AUTHOR]

Sperm hyperactivation is described as a fast whip movement of the flagellum, an irregular trajectory, and an asymmetrically flagellum bend. This motility pattern is achieved during the passage of the sperm along the female genital tract. It helps the spermatozoa to cross through different viscous ambient fluids to finally reach the oocyte. Important signaling proteins are located in the sperm head and flagellum, and they all play an important role in the cascade that controls the sperm hyperactivation. The presence of HCO3− modulates the activity of the soluble adenylyl cyclase (sAC), leading to the production of cAMP. In turn, cAMP modulates the sperm-specific Na+/H+ exchanger (sNHE) and the t-complex protein 11 (TCP11) which play an essential role on the signaling pathway (cAMP/PKA and tyrosine phosphorylation) and sperm hypermotility. sNHE, cystic fibrosis transmembrane conductance regulator (CFTR), and voltage-gated proton channel (Hv) mainly contribute to the regulation of the intracellular pH (pHi) during capacitation. HCO3− entrance and the removal of H+ from the cytoplasm induces the alkalization of pHi, and this change will contribute to the activation of the cation channel of sperm (CatSper). Recently, it was described the participation on sperm motility and the regulation of calcium channels of an autophagy-related protein, the microtubule-associated protein light chain 3 (LC3). This review gathers important literature about the essential roles of sAC, sNHE, CFTR, Hv, and CatSper in the acquisition of sperm hyperactivation, and provides an integrated overview of recently described roles of TCP11 and LC3 on the sperm signaling pathway. Additionally, we provide insight into the infertility induced by the dysfunction of these critical proteins. [ABSTRACT FROM AUTHOR]

Background: Ca2+-signaling controls sperm cell functions necessary for successful fertilization. Multiple endocrine disrupting chemicals have been found to interfere with normal Ca2+-signaling in human sperm cells through an activation of the sperm-specific CatSper Ca2+-channel, which is vital for normal male fertility. Objectives: We investigated 53 pesticides for their ability to interfere with CatSper mediated Ca2+-signaling and function in human sperm cells. Methods: Effects of the pesticides on Ca2+-signaling in human sperm cells were evaluated using a Ca2+-fluorometric assay. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function and viability were assessed using an image cytometry-based acrosome reaction assay and the modified Kremer’s sperm–mucus penetration assay. Results: 28 of 53 pesticides were found to induce Ca2+-signals in human sperm cells at 10 µM. The majority of these 28 active pesticides induced Ca2+-signals through CatSper and interfered with subsequent Ca2+-signals induced by the two endogenous CatSper ligands progesterone and prostaglandin E1. Multiple active pesticides were found to affect Ca2+-mediated sperm functions and viability at 10 µM. Low nM dose mixtures of the active pesticides alone or in combination with other environmental chemicals were found to significantly induce Ca2+-signals and inhibit Ca2+-signals induced subsequently by progesterone and prostaglandin E1. Conclusions: Our results show that pesticides, both alone and in low nM dose mixtures, interfere with normal Ca2+-signaling in human sperm cells in vitro in low nM concentrations. Biomonitoring of the active pesticides in relevant matrices such as blood and reproductive fluids is very limited and the effects of real time human pesticide exposure on human sperm cells and fertility thus remains largely unknown. To which extent human pesticide exposure affects the chances of a successful fertilization in humans in vivo needs further research.

Opposing findings have been published on the regulation of the sperm-specific Ca 2+ channel CatSper (cation channel of sperm) in human sperm cells by the plant triterpenoids lupeol and pristimerin. While the original study on this topic found these triterpenoids to act as potent inhibitors of human CatSper, subsequent studies have failed to replicate such an inhibitory effect. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the studies. The aim of this study was to elucidate this controversy by investigating the batches of lupeol and pristimerin used in our previous study with state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods to reveal potential purity and/or batch variation issues. When comparing the NMR-spectra obtained from 1H-NMR and 13C-NMR with previously published NMR-spectra for lupeol and pristimerin, we could confirm that both the lupeol and pristimerin batch were ≥95 % pure. These results confirm the validity of the findings in our previous study for lupeol and pristimerin, showing that lupeol and pristimerin do not inhibit activation of CatSper in human sperm. In conclusion, using 1H-, 13C- and 2D-NMR methods, we confirm that the lupeol and pristimerin batches used in our previous study were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of lupeol and pristimerin to inhibit activation of CatSper in human sperm.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility. [ABSTRACT FROM AUTHOR]

Background and Purpose: Varicocele is a leading cause of male infertility. Melatonin is a highly pleiotropic neurohormone. We aimed to characterize the melatonin epigenetic potential in varicocele and the involved molecular mechanisms. Experimental Approach Fifty‐two male albino rats were randomly divided into four groups (13 rats each): control (I), melatonin (II), varicocele (III) and melatonin treated varicocele (IV) groups. Left varicocele was induced by partial left renal vein ligation. Reproductive hormones, epididymal sperm functional parameters, testicular 3/17 β‐hydroxysteroid dehydrogenases, antioxidant enzymes, malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase, 8‐hydroxy‐2′‐deoxyguanosine and histopathological/Johnsen's score were evaluated. Flow cytometry and Comet were carried out to explore extent of sperm and testicular DNA damage. Testicular expression of silent information regulator 1 (SIRT1), forkhead transcription factors‐class O (type1) (FOXO1), tumour suppressor gene, P53, cation channels of sperm (CatSper) and steroidogenic acute regulatory protein was evaluated by western blot technique. Testicular expression of Bcl‐2 and its associated X protein and nuclear factor kappa‐light‐chain‐enhancer of activated B cells were assayed by immunohistochemical staining. Testicular miR‐34a expression was quantified by quantitative reverse transcription‐polymerase chain reaction. Key Results: The varicocele induced testicular histological injury, enhanced oxidative stress, P53‐mediated apoptosis, DNA damage and increased testicular miR‐34a expression paralleled with down‐regulated SIRT1/FOXO axis. Melatonin treatment of varicocele rats displayed antioxidant/anti‐apoptotic efficacy and improved reproductive hormones axis, CatSper expression and fertility parameters. MiR‐34a/SIRT1/FOXO1 epigenetic axis integrates testicular melatonin mediated intracellular transduction cascades in varicocele. Conclusion and Implications: Melatonin can be used as an adjuvant therapy to improve varicocele and its complication. [ABSTRACT FROM AUTHOR]

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization.

Opposing findings have been published on the regulation of the sperm-specific Ca2+ channel CatSper (cation channel of sperm) in human sperm cells by the plant triterpenoids lupeol and pristimerin. While the original study on this topic found these triterpenoids to act as potent inhibitors of human CatSper, subsequent studies have failed to replicate such an inhibitory effect. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the studies. The aim of this study was to elucidate this controversy by investigating the batches of lupeol and pristimerin used in our previous study with state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods to reveal potential purity and/or batch variation issues. When comparing the NMR-spectra obtained from 1H-NMR and 13C-NMR with previously published NMR-spectra for lupeol and pristimerin, we could confirm that both the lupeol and pristimerin batch were ≥95 % pure. These results confirm the validity of the findings in our previous study for lupeol and pristimerin, showing that lupeol and pristimerin do not inhibit activation of CatSper in human sperm. In conclusion, using 1H-, 13C- and 2D-NMR methods, we confirm that the lupeol and pristimerin batches used in our previous study were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of lupeol and pristimerin to inhibit activation of CatSper in human sperm.

In mammalian females, after sperm are deposited in the reproductive tract, a fraction of sperm migrates to the lower oviduct (isthmus) and forms a sperm storage site known as the functional sperm reservoir. The interactions between sperm membrane proteins and oviduct epithelial cells facilitate sperm binding to the oviductal epithelium and retention in the reservoir. Sperm are bound by glycans that contain specific motifs present on isthmic epithelial cells. Capacitated sperm are released from the reservoir and travel further in the oviduct to the ampulla where fertilization occurs. For decades, researchers have been studying the molecules and mechanisms of sperm release from the oviductal sperm reservoir. However, it is still not clear if the release of sperm is triggered by changes in sperm, oviduct cells, oviduct fluid, or a combination of these. While there is a possibility that more than one of these events are involved in the release of sperm from the reservoir, one activator of sperm release has the largest accumulation of supporting evidence. This mechanism involves the steroid hormone, progesterone, as a signal that induces the release of sperm from the reservoir. This review gathers and synthesizes evidence for the role of progesterone in inducing sperm release from the oviduct functional sperm reservoir. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) is a strong central nervous system stimulant that has toxic effects on the reproductive system. The aim of this study was to evaluate changes in gene and protein expression levels of CatSper1, 2, and oxidative stress after exposure to METH in the testis of mice. The mRNA and protein expression levels of CatSper1, 2 decreased in the experimental groups. The administration of METH caused a decrease of SOD and CAT activities and an increase in the MDA level. METH damaged the male reproductive system via the oxidative stress pathway and by reduction of CatSper1, 2 genes and protein expression. Mini-abstract: Administration of METH damaged the male reproductive system via the oxidative stress pathway and reduction of CatSper1, 2 genes and protein expression. [ABSTRACT FROM AUTHOR]

Opposing findings have been published on the regulation of the sperm-specific Ca 2+ channel CatSper (cation channel of sperm) in human sperm cells by the plant triterpenoids lupeol and pristimerin. While the original study on this topic found these triterpenoids to act as potent inhibitors of human CatSper, subsequent studies have failed to replicate such an inhibitory effect. It has been suggested that these issues could in part be due to purity issues and/or batch variation between the plant-derived extracts of lupeol and pristimerin obtained for the studies. The aim of this study was to elucidate this controversy by investigating the batches of lupeol and pristimerin used in our previous study with state-of-the-art 1H-, 13C- and 2D-nuclear magnetic resonance (NMR) methods to reveal potential purity and/or batch variation issues. When comparing the NMR-spectra obtained from 1H-NMR and 13C-NMR with previously published NMR-spectra for lupeol and pristimerin, we could confirm that both the lupeol and pristimerin batch were ≥95 % pure. These results confirm the validity of the findings in our previous study for lupeol and pristimerin, showing that lupeol and pristimerin do not inhibit activation of CatSper in human sperm. In conclusion, using 1H-, 13C- and 2D-NMR methods, we confirm that the lupeol and pristimerin batches used in our previous study were ≥95 % pure and thereby fail to identify any purity issues and/or batch variation that could explain the observed inability of lupeol and pristimerin to inhibit activation of CatSper in human sperm.

Study questionAre significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success?Summary answerNegligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF.What is known alreadySperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects.Study design, size and durationSpermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study.Participants/materials, setting, methodsSamples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities.Main results and the role of chancePatch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function.Limitations, reasons for cautionFor practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects.Wider implications of the findingsThese data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized.Study funding/competing interestsThe majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest.Trial registration numberNot applicable.

Study questionAre significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?Summary answerSperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF.What is known alreadyIn human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception.Study design, size, durationSpermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study.Participants/materials, setting, methodsSamples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis.Main results and the role of chanceA total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF.Limitations, reasons for cautionA key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx.Wider implications of the findingsThese data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.

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 longsought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development. [ABSTRACT FROM AUTHOR]

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. • Efficient metagenomics pipeline established for unpurified eukaryote genome assembly. • Two Polymyxa betae genomes were successfully assembled and annotated. • The first P. betae mitochondrial genome is also reported. • Comparison reveals closer relationship between P. betae and Spongospora subterranea. • CatSper gene complex may essential for zoospore motility in plasmodiophorids. [ABSTRACT FROM AUTHOR]

Background and Aim: Genetic changes and mutations in the genes involved in spermatogenesis can occur by environmental and congenital factors. Considering, the extensive use of diazinon in the farms and because we found no study on the effects of diazinon on the expression of CatSper1 gene, a key element of male fertility, we decided to perform this study in order to determine the effects of different doses of diazinon on CatSper1 gene expression, sperm motility and thickness of seminiferous tubules in adult male mice. Materials and Methods: Twenty-four adult male Balb/c mice were randomly divided into four groups. The control group did not receive any injection. The sham group received diazinon solvent. Diazinon1 group received 7.5 mg/kg and, diazinon group 2 received 30 mg/kg of diazinon intraperitoneally, once a day for 2 weeks. After 35 days, we studied the sperms from the molecular and histological aspects and sperm motility was evaluated. Results: Sperm motility was significantly decreased in diazinon 1 (P-value = 0.001) and diazinon 2 (P-value = 0.001) groups compared to that in the control group. Gene expression in diazinon group 1 and diazinone group 2 (P-value = 0.002) was significantly lower than that in the control group. Histological examination also showed that diazinon treatment reduced germinal epithelium thickness and led to vacuole formation and degeneration. Epithelial thickness was significantly decreased in diazinon group 1 (P-value = 0.003) and diazinon group 2 (P-value = 0.006) in comparison to that in the control group. Conclusion: Diazinon affects male fertility by reduction in sperm motility and degenerative changes in germinal epithelium. It also, decreases expression of CatSper1 gene, a key gene in fertility.

Background and Aim:Genetic changes and mutations in the genes involved in spermatogenesis can occur by environmental and congenital factors. Considering, the extensive use of diazinon in the farms and because we found no study on the effects of diazinon on the expression ofCatSper1 gene, a key element of male fertility, we decided to perform this study in order to determine the effects of different doses of diazinon on CatSper1 gene expression, sperm motility and thickness of seminiferous tubules in adult male mice.Materials and Methods:Twenty-four adult male Balb/c mice were randomly divided into four groups. The control group did not receive any injection. The sham group received diazinon solvent. Diazinon1 group received 7.5 mg/kg and, diazinon group 2 received 30 mg/kg of diazinon intraperitoneally, once a day for 2 weeks. After 35 days, we studied the sperms from the molecular and histological aspects and sperm motility was evaluated.Results:Sperm motility was significantly decreased in diazinon 1 (P-value = 0.001) and diazinon 2 (P-value = 0.001) groups compared to that in the control group. Gene expression in diazinon group 1 and diazinone group 2 (P-value = 0.002) was significantly lower than that in the control group. Histological examination also showed that diazinon treatment reduced germinal epithelium thickness and led to vacuole formation and degeneration. Epithelial thickness was significantly decreased in diazinon group 1 (P-value = 0.003) and diazinon group 2 (P-value = 0.006) in comparison to that in the control group.Conclusion:Diazinon affects male fertility by reduction in sperm motility and degenerative changes in germinal epithelium. It also, decreases expression of CatSper1 gene, a key gene in fertility.

Since the molecular similarities and differences among physiological capacitation and cryocapacitation have not been studied in detail, this study was designed to assess the gene and protein expression levels of the Cation channel of sperm (CatSper) 1 and 2, sodium bicarbonate (Na+/HCO3−) cotransporter (NBC) and protein kinase A (PKA) in un-capacitated (control), in vitro capacitated (CAP) and cryopreserved (CRYO) bovine spermatozoa. All samples were subjected to motility evaluation using the computer assisted sperm analysis and chlortetracycline (CTC) assay for the assessment of the capacitation patterns. Furthermore, quantitative reverse transcription PCR (qRT-PCR) and Western blots were used to monitor the expression patterns of the selected capacitation markers. The results showed a significant reduction in the gene and protein expression levels of CatSper1 and 2 in the CRYO group when compared to the CAP group (p < 0.0001). In the case of NBC, the results were not significantly different or were inconclusive. While a non-significant down-regulation of PKA was found in the CRYO group, a significant reduction in the expression of the PKA protein was found in frozen-thawed spermatozoa in comparison to the CAP group (p < 0.05). In conclusion, we may hypothesize that while in vitro capacitated and cryopreserved spermatozoa exhibit CTC-patterns consistent with capacitation events, the molecular machinery underlying CTC-positivity may be different.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.