In Silico Determination of the Effect of Multi-Target Drugs on Calcium Dynamics Signaling Network Underlying Sea Urchin Spermatozoa Motility

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca²⁺]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca²⁺]_i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca²⁺] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na⁺ channels (HCN), [Ca²⁺]-dependent Cl⁻ channels (CaCC) and [Ca²⁺]-dependent K⁺ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca²⁺]-dependent K⁺ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca²⁺] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.     

Zn induces hyperpolarization by activation of a K channel and increases intracellular Ca and pH in sea urchin spermatozoa

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]_i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.     

Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility

Speract, an egg-derived sperm-activating peptide, induces changes in intracellular Ca2+, Na+, pH, cAMP, cGMP, and membrane potential in sperm of the sea urchin Strongylocentrotus purpuratus. Ca2+ is a key regulator of motility in all sperm and, in many marine species, is required for generating turns interspersed with straighter swimming paths that are essential for chemotaxis towards the egg. We show that speract triggers a train of increases in flagellar Ca2+, and that each individual Ca2+ fluctuation induces a transient increase in flagellar asymmetry that leads to a turn. We also find that modifying the amplitude, duration and interval between individual Ca2+ fluctuations by treating sperm with niflumic acid, an inhibitor of Ca2+-activated Cl(-) channels, correspondingly alters the properties of the sperm turns. We conclude that Ca2+ entry through a fast flagellar pathway not only induces sperm turns, but the kinetics of Ca2+ entry may shape the nature of these turns, and that these kinetics are tuned by other channels, possibly including Cl(-) channels. In addition, the speract-induced changes in sperm motility closely resemble those seen during chemotaxis in other marine organisms, yet speract is not a chemoattractant. This implies the Ca2+-induced motility changes are necessary but not sufficient for chemotaxis.     

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca²⁺ ([Ca²⁺]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca²⁺]i before the increase. Both directions of the [Ca²⁺]i change were completely blocked in high K⁺ seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca²⁺]i although both cGMP and cAMP increased the [Ca²⁺]i. The decrease in the [Ca²⁺]i induced by cGMP was more notable following a second photolytic event, once [Ca²⁺]i had been elevated by an initial flash. This pattern of [Ca²⁺]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca²⁺]i decrease is due to a Na⁺/Ca²⁺ exchanger activity, stimulated by hyperpolarization mediated by K⁺ efflux through cGMP-regulated K⁺ channels.     

FLAGELLAR MOTILITY IS NOT INVOLVED IN THE INCORPORATION OF THE SPERM INTO THE EGG AT FERTILIZATION*

Cinemicrography of sea urchin fertilization reveals that the fertilizing sperm is one of the first sperm to attach to the egg. Just before the cortical reaction the fertilizing sperm ceases motility and then is incorporated into the egg without flagellar beating. The rate of incorporation is 5–11 μm/sec and is constant. Lytechinus pictus sperm rendered immotile by azide treatment can bind to and fertilize eggs but binding, and therefore fertilization, is blocked by azide treatment of Strongylocentrotus purpuratus gametes.     

Temporal sampling,resetting, and adaptation orchestrate gradient sensing in sperm

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca²⁺ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca²⁺ response was produced. Additional molecules delivered during a Ca²⁺ response reset the cell by causing a pronounced Ca²⁺ drop that terminated the response; this reset was followed by a new Ca²⁺ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.     

Modulation of the voltage-sensitive Na+/H+ exchange in sea urchin spermatozoa through membrane potential changes induced by the egg peptide speract

Sea urchin sperm motility can be activated by alkalinization of the internal pH, and previous studies have shown that the internal pH can be regulated by a voltage-sensitive Na+/H+ exchanger present in the flagellar plasma membrane. In this study, the effects of speract, a peptide purified from egg conditioned media, on the Na+/H+ exchange were investigated. Evidence presented indicates that speract activates K+ channels in the flagellar membrane and modulates the Na+/H+ exchange activity through resultant changes in membrane potential. In the presence of tetraphenylphosphonium, a lipophilic ion, or high external Na+, the isolated flagella were depolarized, and Na+/H+ exchanger was inhibited. Speract and valinomycin, a K+ ionophore, were able to reactivate 22Na+ uptake, H+ efflux, and alkalinization of intraflagellar pH under either of the depolarizing conditions. Membrane potential measurements using 3,3'-dipropylthiodicarbocyanide iodide indicated repolarization by either speract or valinomycin. The speract-induced voltage changes did not require Na+ but were sensitive to [K+]. Thus, speract induced a slight depolarization in Na+-free seawater with 10 mM K+ but a hyperpolarization with 2 mM K+. Further support for the activation of K+ channels in the flagella was the 2-5-fold stimulation of K+ efflux induced by speract as measured with a K+ electrode. The ionic selectivity of the speract-activated channel assessed by voltage measurements was K+ greater than Rb+ greater than Cs+. The half-maximally effective concentration of speract was about 0.2 nM. That the H+ and K+ efflux in response to peptide was receptor-mediated was confirmed by the use of speract or resact on intact sea urchin spermatozoa, where the peptides were found to stimulate K+ efflux and to reverse the tetraphenylphosphonium inhibition on H+ efflux only in the homologous spermatozoa. Modulation of the voltage-sensitive Na+/H+ exchange by egg peptides, therefore, appears to be indirect and is coupled through its action on membrane potential.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

Inverse relationship of Ca<Superscript>2+</Superscript>-dependent flagellar response between animal sperm and prasinophyte algae

Symmetry/asymmetry conversion of eukaryotic flagellar waveform is caused by the changes in intracellular Ca²⁺. Animal sperm flagella show symmetric or asymmetric waveform at lower or higher concentration of intracellular Ca²⁺, respectively. In Chlamydomonas, high Ca²⁺ induces conversion of flagellar waveform from asymmetric to symmetry, resulting in the backward movement. This mirror image relationship between animal sperm and Chlamydomonas could be explained by the distinct calcium sensors used to regulate the outer arm dyneins (Inaba 2015). Here we analyze the flagellar Ca²⁺-response of the prasinophyte Pterosperma cristatum, which shows backward movement by undulating four flagella, the appearance similar to animal sperm. The moving path of Pterosperma shows relatively straight in artificial seawater (ASW) or ASW in the presence of a Ca²⁺ ionophore A23187, whereas it becomes circular in a low Ca²⁺ solution. Analysis of flagellar waveform reveals symmetric or asymmetric waveform propagation in ASW or a low Ca²⁺ solution, respectively. These patterns of flagellar responses are completely opposite to those in sperm flagella of the sea urchin Anthocidaris crassispina, supporting the idea previously proposed that the difference in flagellar response to Ca²⁺ attributes to the evolutional innovation of calcium sensors of outer arm dynein in opisthokont or bikont lineage.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.     

Regulation of spermatozoa motility in response to cations in Russian sturgeon Acipenser gueldenstaedtii

The aim of this study was to investigate the response of Russian sturgeon (Acipenser gueldenstaedtii) sperm to external cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) and their susceptibility on the induction of motility and swimming behavior. An in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. Sperm motility was inhibited by 60 mm NaCl (∼140 mOsm/kg) and 0.7 mm KCl solutions (∼ 21.4 mOsm/kg). The Ca²⁺ and Mg²⁺ ions were not able to inhibit spermatozoa motility. By contrast, Na⁺ within a limited concentration range (between 45 and 55 mm) was able to reverse the inhibitory effect of K⁺ at the critical concentration (0.7 mm). Ca²⁺ and Mg²⁺ were also able to reverse the K⁺-mediated spermatozoa motility restriction at concentrations starting at 0.01 and 0.1 mm, respectively. These results provide evidence for the role of K⁺ in suppressing spermatozoa motility, and suggest that Ca²⁺, Mg²⁺, and possibly Na⁺ trigger motility in Russian sturgeon sperm.     

Certain Strongylocentrotus purpuratus sperm mitochondrial proteins co-purify with low density detergent-insoluble membranes and are PKA or PKC-substrates possibly involved in sperm motility regulation

Background

Sea urchin sperm motility is regulated by Speract, a sperm-activating peptide (SAP) secreted from the outer egg coat. Upon binding to its receptor in the sperm flagellum, Speract induces a series of ionic and metabolic changes in Strongylocentrotus purpuratus spermatozoa that regulate their motility. Among these events, protein phosphorylation is one of the most relevant and evidence indicates that some proteins of the Speract signaling cascade localize in low density detergent-insoluble membranes (LD-DIM).

Methods

LD-DIM-derived proteins from immotile, motile or Speract-stimulated S. purpuratus sperm were resolved in 2-D gels and the PKA and PKC substrates detected with specific antibodies were identified by LC–MS/MS.

Results

Differential PKA and PKC substrate phosphorylation levels among the LD-DIM isolated from sperm in different motility conditions were found and identified by mass spectrometry as: ATP synthase, creatine kinase, NADH dehydrogenase (ubiquinone) flavoprotein 2, succinyl-CoA ligase and the voltage-dependent anion channel 2 (VDAC2), which are mitochondrial proteins, as well as, the cAMP-dependent protein kinase type II regulatory (PKA RII) subunit, Tubulin β chain and Actin Cy I changed their phosphorylation state.

Conclusions

Some mitochondrial proteins regulated by PKA or PKC may influence sea urchin sperm motility.

General significance

The fact that a high percentage (66%) of the PKA or PKC substrates identified in LD-DIM are mitochondrial proteins suggests that the phosphorylation of these proteins modulates sea urchin sperm motility via Speract stimulation by providing sufficient energy to sperm physiology. Those mitochondrial proteins are indeed PKA- or PKC-substrates in the sea urchin spermatozoa.     

Intracellular signal transduction pathways in the regulation of fowl sperm motility: Evidence for the involvement of phosphatidylinositol 3‐kinase (PI3‐K) cascade

The possible role of PI3‐K in the reversible temperature‐dependent immobilization of fowl sperm motility was investigated by using PI3‐K inhibitor (LY294002) and its inactive analogue (LY303511). The existence of the PI3‐K in fowl spermatozoa was also confirmed by Western blotting analysis. Fowl sperm motility in TES/NaCl buffer remained negligible at the avian body temperature of 40°C but was maintained vigorously when the temperature was decreased to 30°C. At 30°C, no stimulation or inhibition of motility was observed after the addition of 2 mM CaCl₂ and 10 µM LY294002 or LY303511: around 70–80% of spermatozoa remained motile. In contrast, at 40°C, the motility of spermatozoa was activated immediately after the addition of Ca²⁺, but the subsequent addition of LY294002 inhibited the motility again. The addition of LY303511 did not appreciably affect the Ca²⁺‐supplemented sperm motility, which was maintained for at least 15 min. The ATP concentrations of spermatozoa after the addition of LY294002 + Ca²⁺ or LY303511 + Ca²⁺ were almost the same values compared with those of Ca²⁺ alone at 40°C, suggesting that the addition of LY294002 was not simply affecting membrane damage or inhibiting energy production in the spermatozoa, but may be acting on some part of the motility‐regulating cascade. Immunoblotting of sperm extract using an antibody to PI3‐K revealed a major cross‐reacting protein of 85 kDa, which corresponds to the molecular weight of the subunit of PI3‐K. These results suggest that PI3‐K may be positively involved in the calcium‐regulated maintenance of flagellar movement of fowl spermatozoa at 40°C. Mol. Reprod. Dev. 76: 603–610, 2009. © 2008 Wiley‐Liss, Inc.     

Huntingtin regulates Ca chemotaxis and K-facilitated cAMP chemotaxis,in conjunction with the monovalent cation/H exchanger Nhe1, in a model developmental system: Insights into its possible role in Huntington׳s disease

Huntington?s disease is a neurodegenerative disorder, attributable to an expanded trinucleotide repeat in the coding region of the human HTT gene, which encodes the protein huntingtin. These mutations lead to huntingtin fragment inclusions in the striatum of the brain. However, the exact function of normal huntingtin and the defect causing the disease remain obscure. Because there are indications that huntingtin plays a role in Ca²⁺ homeostasis, we studied the deletion mutant of the HTT ortholog in the model developmental system Dictyostelium discoideum, in which Ca²⁺ plays a role in receptor-regulated behavior related to the aggregation process that leads to multicellular morphogenesis. The D. discoideum htt⁻-mutant failed to undergo both K⁺-facilitated chemotaxis in spatial gradients of the major chemoattractant cAMP, and chemotaxis up a spatial gradient of Ca²⁺, but behaved normally in Ca²⁺-facilitated cAMP chemotaxis and Ca²⁺-dependent flow-directed motility. This was the same phenotypic profile of the null mutant of Nhel, a monovalent cation/H⁺exchanger. The htt⁻-mutant also failed to orient correctly during natural aggregation, as was the case for the Nhel mutant. Moreover, in a K⁺-based buffer the normal localization of actin was similarly defective in both htt⁻ and nhe1⁻ cells in a K⁺-based buffer, and the normal localization of Nhe1 was disrupted in the htt⁻ mutant. These observations demonstrate that Htt and Nhel play roles in the same specific cation-facilitated behaviors and that Nhel localization is directly or indirectly regulated by Htt. Similar cation-dependent behaviors and a similar relationship between Htt and Nhe1 have not been reported for mammalian neurons and deserves investigation, especially as it may relate to Huntington?s disease.     

Sperm biology and control of reproduction in sturgeon: (II) sperm morphology, acrosome reaction, motility and cryopreservation

In the present review, sperm morphology, acrosome reaction, motility, short-term storage and cryopreservation are summarized and discussed in sturgeon (Chondrostei, Acipenseriformes). The elongated head of spermatozoon comprises an acrosome with 8?C12 posterolateral projections. Usually three endonuclear canals are observed in the nucleus. Proximal and distal centrioles and 3?C6 mitochondria are located in the midpiece region. The flagellum consists of an axoneme with a typical ??9?+?2?? structure of microtubules and presents a ribon-like structure due to two lateral membranous fins. Egg water, Ca²⁺ and Mg²⁺ can trigger acrosome reaction. Trypsin- and chymotrypsin-like activities are reported in sturgeon sperm. These physiological properties of sturgeon sperm are identified as serine activity with 33?kDa molecular mass and can be inhibited by their respective inhibitors. The K⁺ prevents sperm activation in seminal plasma, and hypo-osmolality or decrease of extracellular K⁺ triggers sperm activation. Extracellular Ca²⁺ is involved in flagellar beating pattern and sperm velocity. After activation, sperm motility, velocity, and flagellar beating frequency, wavelength and amplitude decrease, while number of waves and curvature increase. Sturgeon sperm can be stored for several days at 4?°C; however it is better to add K⁺ into the immobilizing medium because it prevents sperm activation during incubation. Regarding sperm cryopreservation, methanol is a better cryoprotectant than DMSO. Either short-term storage or cryopreservation of sperm generates damage to spermatozoa that lead to reduction of sperm motility performance. Some studies suggest using an activation medium containing Ca²⁺ for enhancing sperm motility performance of incubated or frozen-thawed sperm.     

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca²⁺ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca²⁺ oscillation without changing the basal intracellular Ca²⁺ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca²⁺ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca²⁺ regulation may be a common feature among animal sperm.     

What Is the Core Oscillator in the Speract-Activated Pathway of the Strongylocentrotus purpuratus Sperm Flagellum?

Sperm chemotaxis has an important role in fertilization. Most of our knowledge regarding this phenomenon comes from studies in organisms whose fertilization occurs externally, like sea urchins. Sea urchin spermatozoa respond to sperm-activating peptides, which diffuse from the egg jelly coat and interact with their receptor in the flagellum, triggering several physiological responses: changes in membrane potential, intracellular pH, cyclic nucleotide levels, and intracellular Ca2+ concentration ([Ca2+]). In particular, flagellar [Ca2+] has been shown to oscillate. These [Ca2+] oscillations are correlated with changes in the flagellar shape and so with the regulation of the sperm swimming paths. In this study, we demonstrate, from a mathematical modeling perspective, that the reported speract-activated signaling pathway in Strongylocentrotus purpuratus (speract being a sperm-activating peptide specific to this species) has the necessary elements to replicate the reported [Ca2+] oscillations. We further investigate which elements of this signaling pathway constitute the core oscillator.