Hamster spermatozoa incorporate hypotaurine via TauT for self-protection.

In brief: Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. Abstract: To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. ß-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.

Hamster Sperm Possess Functional Na+/Ca2+-Exchanger 1: Its Implication in Hyperactivation.

Previous studies demonstrated that hamster sperm hyperactivation is suppressed by extracellular Na+ by lowering intracellular Ca2+ levels, and Na+/Ca2+-exchanger (NCX) specific inhibitors canceled the suppressive effects of extracellular Na+. These results suggest the involvement of NCX in the regulation of hyperactivation. However, direct evidence of the presence and functionality of NCX in hamster spermatozoa is still lacking. This study aimed to reveal that NCX is present and is functional in hamster spermatozoa. First, NCX1 and NCX2 transcripts were detected via RNA-seq analyses of hamster testis mRNAs, but only the NCX1 protein was detected. Next, NCX activity was determined by measuring the Na+-dependent Ca2+ influx using the Ca2+ indicator Fura-2. The Na+-dependent Ca2+ influx was detected in hamster spermatozoa, notably in the tail region. The Na+-dependent Ca2+ influx was inhibited by the NCX inhibitor SEA0400 at NCX1-specific concentrations. NCX1 activity was reduced after 3 h of incubation in capacitating conditions. These results, together with authors' previous study, showed that hamster spermatozoa possesses functional NCX1 and that its activity was downregulated upon capacitation to trigger hyperactivation. This is the first study to successfully reveal the presence of NCX1 and its physiological function as a hyperactivation brake.

Activation of cAMP-dependent phosphorylation pathways is independent of ROS production during mouse sperm capacitation.

Mammalian sperm have to undergo capacitation to fertilize the egg. At the molecular level, capacitation involves cAMP synthesis, protein kinase A activation, and downstream increase in tyrosine phosphorylation. In addition, during capacitation, mammalian sperm actively generate reactive oxygen species (ROS). It has been proposed that ROS modulate phosphorylation pathways; however, the crosstalk between these signaling processes is not well-understood. In the present study, we used loss- and gain-of-function approaches to evaluate the interconnection between ROS and phosphorylation. We showed that BSA and HCO3- , but not Ca2+ , in the capacitation media are required for ROS production. The synergic effect of these compounds was neither mediated by HCO3- stimulation of cAMP synthesis nor by BSA-induced cholesterol efflux. The capacitation-induced ROS generation was blocked in the presence of superoxide dismutase (SOD), catalase, and apocynin. However, none of these compounds affected cAMP-dependent or tyrosine phosphorylation. On the other hand, the addition of NADPH to the media induced ROS generation in sperm incubated in the absence of BSA and HCO3- without upregulating cAMP-dependent or tyrosine phosphorylation signaling. Most interestingly, catalase, but not SOD, blocked in vitro fertilization suggesting a role for H2 O2 in this process.

Oviductal high concentration of K+ suppresses hyperpolarization but does not prevent hyperactivation, acrosome reaction and in vitro fertilization in hamsters.

Mammalian sperm have to undergo capacitation to be fertilization competent. Capacitated sperm in vitro show hyperpolarization of the membrane potential. It has been reported that in mouse membrane hyperpolarization is necessary for the acrosome reaction. We recently found that the fluid of the hamster oviduct, where fertilization occurs, contained a high potassium (K+) concentration (~20 mEq/l). This high K+ concentration could depolarize the membrane potential and prevent the acrosome reaction/fertilization. Conversely, some beneficial effects on capacitation of high K+ concentration or a high K/Na ratio were also reported. In the present study, we investigated the effects of oviduct high K+ concentration on hamster sperm capacitation-associated events and fertilization. The present study confirmed that, in hamster sperm, membrane potential was hyperpolarized upon in vitro capacitation, indicating that capacitation-associated hyperpolarization is a universal phenomenon among mammalian species. An increase in KCl concentration in the medium to 20 mM significantly depolarized the membrane potential and suppressed hyperpolarization when in the presence of >101 mM NaCl. However, an increase in the KCl concentration to 20 mM did not significantly affect the percentage of motile sperm, hyperactivation or the acrosome reaction. No effect of 20 mM KCl on in vitro fertilization was observed. In addition, no correlative changes in hyperactivation and the acrosome reaction with K/Na ratio were observed. These results suggested that in hamsters the oviduct K+ concentration suppressed hyperpolarization but had no effect on capacitation and in vitro fertilization. Our results raised a question over the physiological significance of capacitation-associated hyperpolarization.

Na+/K+-ATPase α4 regulates sperm hyperactivation while Na+/K+-ATPase α1 regulates basal motility in hamster spermatozoa.

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na+. Two types of catalytic Na+/K+-ATPase (NKA) α subunits (α1 and α4) are present in spermatozoa. In this work, the contribution of these NKA subunits to the regulation of hamster sperm hyperactivation was investigated using the specific inhibitor ouabain. When 10-6 M ouabain was added to the modified Tyrode's albumin lactate pyruvate medium (mTALP) medium, hyperactivation was significantly inhibited, whereas 10-5-10-4 M ouabain was needed to significantly reduce the amount of motile spermatozoa. When a more detailed analysis of flagellar movement was performed, 10-6 M ouabain suppressed the hyperactivation-associated change in the patterns of flagellar motion without affecting the sliding velocity of microtubules. Since 10-6 M ouabain specifically inhibits the α4 subunit while 10-5-10-4 M ouabain inhibits both the α1 and α4 subunits, these results suggest the α1 subunit is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. Ouabain did not inhibit tyrosine phosphorylation, and activation of tyrosine phosphorylation-dependent signaling had no effect on the inhibition of hyperactivation by ouabain. The immediate recovery of hyperactivation was observed when ouabain was washed out after a 3-h incubation. whereas the administration of ouabain after the onset of hyperactivation significantly inhibited hyperactivation. These results suggest ouabain inhibited hyperactivation in a manner that was independent of time-requiring phosphorylation-mediated signaling.

Regulatory mechanisms of sperm flagellar motility by metachronal and synchronous sliding of doublet microtubules.

STUDY QUESTION: What is the role of metachronal and synchronous sliding in sperm flagellar motility? SUMMARY ANSWER: Both metachronal and oscillatory synchronous sliding are essential for sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding of a specific pair of the doublet microtubules and the oscillatory synchronous sliding between most pairs of doublet microtubules modulates the sperm flagellar motility. WHAT IS KNOWN ALREADY: Metachronal and synchronous sliding of doublet microtubules are involved in sperm flagellar motility and regulation of these sliding movements controls flagellar bend formation. STUDY DESIGN, SIZE, DURATION: To study the regulatory mechanisms of metachronal and synchronous sliding in flagellar movement of golden hamster spermatozoa, changes in these sliding movements during hyperactivation were examined by measuring the angle of the tangent to the flagellar shaft with reference to the central axis of the sperm head (the shear angle) along the flagellum. Golden hamster spermatozoa were obtained from the caudal epididymis of five sexually mature golden hamsters. Results from three experiments were averaged. The number of spermatozoa analyzed is 15 activated sperm, 22 hyperactivated sperm and 20 acrosome-reacted sperm. PARTICIPANTS/MATERIALS, SETTING, METHODS: For detailed field-by-field analysis, an individual flagellar image was tracked automatically using the Autotrace module of image analysis software. The coordinate values of the flagellar shaft were used to calculate the shear angle, which is proportional to the amount of microtubule sliding at any given position along the flagellum. The maximum shear angles of metachronal and synchronous sliding were obtained from the mean shear angles between the maximum shear angles of pro-hook bends and the absolute values of the minimum shear angles of anti-hook bends, which represent the amplitude of a set of successive shear angle curves, with 3-12 shear curves covering one beat cycle of sperm flagellar movement. Asymmetry of flagellar waves was expressed by the mean shear angle between the maximum shear angle of pro-hook bends and the minimum shear angle of anti-hook bends at 100 µm from the head-midpiece junction. MAIN RESULTS AND THE ROLE OF CHANCE: The asymmetrical flagellar movements observed in the activated (non-hyperactivated) and hyperactivated spermatozoa were characterized by the non-oscillatory synchronous sliding of a specific pair of the doublets; the large asymmetrical flagellar movement in the hyperactivated spermatozoa was generated by the large non-oscillatory synchronous sliding. Both the metachronal and synchronous sliding increased during the hyperactivation; however, the large symmetrical flagellar movement of the acrosome-reacted spermatozoa was characterized by the oscillatory synchronous sliding between most pairs of doublets. These results demonstrated that the metachronal and synchronous sliding are involved in generation and modulation of sperm flagellar motility; however, two types of synchronous sliding, non-oscillatory and oscillatory sliding, modulate the sperm flagellar motility by enhancing the sliding of a specific pair of the doublets or the sliding between most pairs of the doublets. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: This is an indirect study of the metachronal and synchronous sliding of doublet microtubules. Studies based on the direct observation of behavior of dynein are needed to clarify the sliding microtubule theory of flagellar movement of spermatozoa. WIDER IMPLICATIONS OF THE FINDINGS: Both the metachronal and oscillatory synchronous sliding of doublet microtubule generate and modulate sperm flagellar motility, while the change in mode of synchronous sliding between the non-oscillatory synchronous sliding and oscillatory synchronous sliding modulates the sperm flagellar motility. The coordination between these sliding leads to various types of flagellar and ciliary motility, including the asymmetrical beating in flagellar and ciliary movement and planar or helical beating in sea urchin spermatozoa. Moreover, the finding that the metachronal sliding and two types of synchronous sliding generate and modulate the flagellar motility will open a new avenue for quantitative analysis of flagellar and ciliary motility. STUDY FUNDING AND COMPETING INTEREST(S): The authors have no conflict of interest and no funding to declare.

γ-Aminobutyric acid suppresses enhancement of hamster sperm hyperactivation by 5-hydroxytryptamine.

Sperm hyperactivation is regulated by hormones present in the oviduct. In hamsters, 5-hydroxytryptamine (5HT) enhances hyperactivation associated with the 5HT2 receptor and 5HT4 receptor, while 17ß-estradiol (E2) and γ-aminobutyric acid (GABA) suppress the association of the estrogen receptor and GABAA receptor, respectively. In the present study, we examined the regulatory interactions among 5HT, GABA, and E2 in the regulation of hamster sperm hyperactivation. When sperm were exposed to E2 prior to 5HT exposure, E2 did not affect 5HT-enhanced hyperactivation. In contrast, GABA partially suppressed 5HT-enhanced hyperactivation when sperm were exposed to GABA prior to 5HT. GABA suppressed 5HT-enhanced hyperactivation associated with the 5HT2 receptor although it did not suppress 5HT-enhanced hyperactivation associated with the 5HT4 receptor. These results demonstrate that hamster sperm hyperactivation is regulated by an interaction between the 5HT2 receptor-mediated action of 5HT and GABA.

Regulation of hamster sperm hyperactivation by extracellular Na.

Mammalian sperm motility has to be hyperactivated to be fertilization-competent. Hyperactivation is regulated by extracellular environment. Osmolality of mammalian semen is higher than that in female reproductive tract; however, the effect of them on hyperactivation has not been investigated. So we investigated the effect of osmotic environment on hyperactivation using hamster spermatozoa at first. Increase in the osmolality of the media (∼370 mOsm) by increasing the concentration of NaCl (∼150 mmol/L) caused the delay of the expression of hyperactivation. When NaCl concentration varied in the same range (75-150 mmol/L) whereas the osmolality was fixed at 370 mOsm by adding mannitol, the delay of hyperactivation occurred dependent on NaCl concentration. Increase in NaCl concentration also caused suppression of curvilinear velocity, bend angle, and sliding velocity of the flagellum at the onset of incubation, suggesting that NaCl concentration affect both activation and hyperactivation in hamster spermatozoa. Hamster sperm intracellular Ca(2+) concentration decreased as extracellular NaCl concentration increased, whereas membrane potential and intracellular pH were unaffected by extracellular NaCl concentration. SN-6 and SEA0400, inhibitors of Na(+)-Ca(2+) exchanger (NCX), increased intracellular Ca(2+) and accelerated hyperactivation in the presence of 150 mmol/L NaCl. Tyrosine phosphorylation on fibrous sheath proteins was unaffected by extracellular NaCl concentration. These results suggest that extracellular Na(+) suppresses hamster sperm hyperactivation by reducing intracellular Ca(2+) via an action of NCX in a tyrosine phosphorylation-independent manner. It seems that the removal of suppression by extracellular Na(+) leads to the expression of hyperactivated motility.

Non-genomic regulation and disruption of spermatozoal in vitro hyperactivation by oviductal hormones.

During capacitation, motility of mammalian spermatozoon is changed from a state of "activation" to "hyperactivation." Recently, it has been suggested that some hormones present in the oviduct are involved in the regulation of this hyperactivation in vitro. Progesterone, melatonin, and serotonin enhance hyperactivation through specific membrane receptors, and 17ß-estradiol suppresses this enhancement by progesterone and melatonin via a membrane estrogen receptor. Moreover, γ-aminobutyric acid suppresses progesterone-enhanced hyperactivation through the γ-aminobutyric acid receptor. These hormones dose-dependently affect hyperactivation. Although the complete signaling pathway is not clear, progesterone activates phospholipase C and protein kinases and enhances tyrosine phosphorylation. Moreover, tyrosine phosphorylation is suppressed by 17ß-estradiol. This regulation of spermatozoal hyperactivation by steroids is also disrupted by diethylstilbestrol. The in vitro experiments reviewed here suggest that mammalian spermatozoa are able to respond to effects of oviductal hormones. We therefore assume that the enhancement of spermatozoal hyperactivation is also regulated by oviductal hormones in vivo.

Estrogen suppresses melatonin-enhanced hyperactivation of hamster spermatozoa.

Hamster sperm hyperactivation is enhanced by progesterone, and this progesterone-enhanced hyperactivation is suppressed by 17ß-estradiol (17ßE2) and γ-aminobutyric acid (GABA). Although it has been indicated that melatonin also enhances hyperactivation, it is unknown whether melatonin-enhanced hyperactivation is also suppressed by 17ßE2 and GABA. In the present study, melatonin-enhanced hyperactivation was significantly suppressed by 17ßE2 but not by GABA. Moreover, suppression of melatonin-enhanced hyperactivation by 17ßE2 occurred through non-genomic regulation via the estrogen receptor (ER). These results suggest that enhancement of hyperactivation is regulated by melatonin and 17ßE2 through non-genomic regulation.

Suppression of progesterone-enhanced hyperactivation in hamster spermatozoa by γ-aminobutyric acid.

It has been recently shown that mammalian spermatozoa were hyperactivated by steroids, amines and amino acids. In the present study, we investigated whether hyperactivation of hamster sperm is regulated by progesterone (P) and γ-aminobutyric acid (GABA). Although sperm hyperactivation was enhanced by P, GABA significantly suppressed P-enhanced hyperactivation in a dose-dependent manner. Suppression of P-enhanced hyperactivation by GABA was significantly inhibited by an antagonist of the GABAA receptor (bicuculline). Moreover, P bound to the sperm head, and this binding was decreased by GABA. Because the concentrations of GABA and P change in association with the estrous cycle, these results suggest that GABA and P competitively regulate the enhancement of hyperactivation through the GABAA receptor.

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm.

Many studies have been conducted to elucidate the relationship between energy metabolic pathways (glycolysis and respiration) and flagellar motility in mammalian sperm, but the contribution of glycolysis to sperm motility has not yet been fully elucidated. In the present study, we performed detailed analysis of mouse sperm flagellar motility for further understanding of the contribution of glycolysis to mammalian sperm motility. Mouse sperm maintained vigorous motility in the presence of substrates either for glycolysis or for respiration. By contrast, inhibition of glycolysis by alpha-chlorohydrine caused a significant decrease in the bend angle of the flagellar bending wave, sliding velocity of outer doublet microtubules and ATP content even in the presence of respiratory substrates (pyruvate or ß-hydroxybutyrate). The decrease of flagellar bend angle and sliding velocity are prominent in the distal part of the flagellum, indicating that glycolysis inhibition caused the decrease in ATP concentration threrein. These results suggest that glycolysis potentially acts as a spatial ATP buffering system, transferring energy (ATP) synthesized by respiration at the mitochondria located in the basal part of the flagellum to the distal part. In order to validate that glycolytic enzymes can transfer high energy phosphoryls, we calculated intraflagellar concentration profiles of adenine nucleotides along the flagellum by computer simulation analysis. The result demonstrated the involvement of glycolysis for maintaining the ATP concentration at the tip of the flagellum. It is likely that glycolysis plays a key role in energy homeostasis in mouse sperm not only through ATP production but also through energy transfer.

Transient Ca2+ mobilization caused by osmotic shock initiates salmonid fish sperm motility.

Salmonid fish sperm motility is known to be suppressed in millimolar concentrations of extracellular K(+), and dilution of K(+) upon spawning triggers cAMP-dependent signaling for motility initiation. In a previous study, however, we demonstrated that suspending sperm in a 10% glycerol solution and subsequent dilution into a low-osmotic solution induced motility independently of extracellular K(+) and cAMP. In the present study, we further investigated the glycerol-induced motility mechanism. We found that treatment with solutions consisting of organic or inorganic ions, as well as glycerol, induced sperm motility in an osmolarity-dependent manner. Elimination of intracellular Ca(2+) by BAPTA-AM significantly inhibited glycerol-treated sperm motility, whereas removal of extracellular Ca(2+) by EGTA did not. Monitoring intracellular Ca(2+), using fluo-4, revealed that intracellular Ca(2+) increased when sperm were suspended in hypertonic solutions, and a subsequent dilution into a hypotonic solution led to a decrease in intracellular Ca(2+) concomitant with motility initiation. In addition, upon dilution of sperm into a hypertonic glycerol solution prior to demembranation, the motility of demembranated sperm was reactivated in the absence of cAMP. The motility recovery suggests that completion of axonemal maturation occurred during exposure to a hypertonic environment. As a result, it is likely that glycerol treatment of sperm undergoing hypertonic shock causes mobilization of intracellular Ca(2+) from the intracellular Ca(2+) store and also causes maturation of axonemal proteins for motility initiation. The subsequent dilution into a hypotonic solution induces a decrease in intracellular Ca(2+) and flagellar movement. This novel mechanism of sperm motility initiation seems to act in a salvaging manner for the well-known K(+)-dependent pathway.