Sea Urchin Sperm Research Articles

Mitochondrial permeability transition pore (MPTP) desensitization increases sea urchin spermatozoa fertilization rate.

Mitochondrial permeability transition pore (MPTP) is a protein complex whose opening promotes an abrupt increase in mitochondrial inner membrane permeability. Calcium signaling pathways are described in gametes and are involved in the fertilization process. Although mitochondria may act as Ca(2+) store and have a fast calcium-releasing mechanism through MPTP, its contribution to fertilization remains unclear. The work aimed to investigate the MPTP phenomenon in sea urchin spermatozoa and its role on the fertilization. Several pharmacological tools were used to evaluate the MPTP's physiology. Our results demonstrated that MPTP occurs in male gametes in a Ca(2+) - and voltage-dependent manner and it is sensitive to cyclosporine A. Additionally, our data show that MPTP opening does not alter ROS generation in sperm cells. Inhibition of MPTP in spermatozoa strongly improved the fertilization rate, which may involve mechanisms that increase the spermatozoa lifespan. The present work is the first report of the presence of a voltage- and Ca(2+) -dependent MPTP in gametes of invertebrates and indicates MPTP opening as another evolutionary feature shared by sea urchins and mammals. Studies about MPTP in sea urchin male gametes may contribute to the elucidation of several mechanisms involved in sperm infertility.

Mechanics of the eukaryotic flagellar axoneme: Evidence for structural distortion during bending.

The sliding doublet mechanism is the established explanation that allows us to understand the process of ciliary and flagellar bending. In this study, we apply the principles of the sliding doublet mechanism to analyze the mechanics of the counterbend phenomenon in sea urchin sperm flagella. When a passive, vanadate-treated, flagellum is forced into a bend with a glass microprobe, the portion of the flagellum distal to the probe exhibits a bend of opposite curvature (counterbend) to the imposed bend. This phenomenon was shown to be caused by the induction of inter-doublet shear and is dependent on the presence of an inter-doublet shear resistance. Here we report that in sea urchin flagella there is systematically less shear induced in the distal flagellum than is predicted by the sliding doublet mechanism, if we follow the assumption that the diameter of the flagellum is uniform. To account for the reduced shear that is observed, the likeliest and most direct interpretation is that the portion of the axoneme that is forced to bend undergoes substantial compression of the axoneme in the bending plane. A compression of 30-50 nm would be sufficient to account for the shear reduction from a bend of 2 radians. A compression of this magnitude would require considerable flexibility in the axoneme structure. This would necessitate that the radial spokes and/or the central pair apparatus are easily compressed by transverse stress. © 2016 Wiley Periodicals, Inc.

Comparative toxicities of selected rare earth elements: Sea urchin embryogenesis and fertilization damage with redox and cytogenetic effects

BackgroundBroad-ranging adverse effects are known for rare earth elements (REE), yet only a few studies tested the toxicity of several REE, prompting studies focusing on multi-parameter REE toxicity. MethodsTrichloride salts of Y, La, Ce, Nd, Sm, Eu and Gd were tested in Paracentrotus lividus sea urchin embryos and sperm for: (1) developmental defects in either REE-exposed larvae or in the offspring of REE-exposed sperm; (2) fertilization success; (3) mitotic anomalies in REE-exposed embryos and in the offspring of REE-exposed sperm, and (4) reactive oxygen species (ROS) formation, and malondialdehyde (MDA) and nitric oxide (NO) levels. ResultsREEs affected P. lividus larvae with concentration-related increase in developmental defects, 10−6 to 10−4M, ranking as: Gd(III)>Y(III)>La(III)>Nd(III)≅Eu(III)>Ce(III)≅Sm(III). Nominal concentrations of REE salts were confirmed by inductively coupled plasma mass spectrometry (ICP-MS). Significant increases in MDA levels, ROS formation, and NO levels were found in REE-exposed embryos. Sperm exposure to REEs (10−5 to 10−4M) resulted in concentration-related decrease in fertilization success along with increase in offspring damage. Decreased mitotic activity and increased aberration rates were detected in REE-exposed embryos and in the offspring of REE-exposed sperm. ConclusionREE-associated toxicity affecting embryogenesis, fertilization, cytogenetic and redox endpoints showed different activities of tested REEs. Damage to early life stages, along with redox and cytogenetic anomalies should be the focus of future REE toxicity studies.

Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism

Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca2+ concentration ([Ca2+]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca2+]i in an external Ca2+ concentration ([Ca2+]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca2+]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca2+ and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca2+]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.

A sea urchin cell-free system to study male pronuclear assembly and activation.

Typically sperm nuclei are genetically inert and contain extremely compacted chromatin. Following fertilization, the first steps in their conversion to somatic nuclei (male pronuclei) which will support further development involve chromatin decondensation and the formation of a new nuclear envelope. We have studied the reactivation of sea urchin sperm nuclei in a cell-free system derived from homogenates of activated sea urchin egg cytoplasm. The cell-free system has provided several novel insights including requirements for sperm-specific histone phosphorylation on N- and C-terminal extensions and disassembly of the sperm nuclear lamina for decondensation, the utilization of remnant regions of the sperm nuclear envelope to direct polarized binding and fusion of egg membranes to form the new nuclear envelope, and a role for phosphoinositide metabolism in initiation of membrane fusion through binding of a minor membrane fraction enriched in PtdIns (4,5)P2, PLCγ and SFK1 which locally produces a fusigenic lipid, diacylglycerol.

A K(+)-selective CNG channel orchestrates Ca(2+) signalling in zebrafish sperm.

Calcium in the flagellum controls sperm navigation. In sperm of marine invertebrates and mammals, Ca(2+) signalling has been intensely studied, whereas for fish little is known. In sea urchin sperm, a cyclic nucleotide-gated K(+) channel (CNGK) mediates a cGMP-induced hyperpolarization that evokes Ca(2+) influx. Here, we identify in sperm of the freshwater fish Danio rerio a novel CNGK family member featuring non-canonical properties. It is located in the sperm head rather than the flagellum and is controlled by intracellular pH, but not cyclic nucleotides. Alkalization hyperpolarizes sperm and produces Ca(2+) entry. Ca(2+) induces spinning-like swimming, different from swimming of sperm from other species. The "spinning" mode probably guides sperm into the micropyle, a narrow entrance on the surface of fish eggs. A picture is emerging of sperm channel orthologues that employ different activation mechanisms and serve different functions. The channel inventories probably reflect adaptations to species-specific challenges during fertilization.

ATP Consumption of Eukaryotic Flagella Measured at a Single-Cell Level

The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural, and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. In this study, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axoneme’s ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with ∼2.3 × 105 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights, to our knowledge, into the beating mechanism of flagella and a powerful tool for future studies.

Sperm navigation along helical paths in 3D chemoattractant landscapes.

Sperm require a sense of direction to locate the egg for fertilization. They follow gradients of chemical and physical cues provided by the egg or the oviduct. However, the principles underlying three-dimensional (3D) navigation in chemical landscapes are unknown. Here using holographic microscopy and optochemical techniques, we track sea urchin sperm navigating in 3D chemoattractant gradients. Sperm sense gradients on two timescales, which produces two different steering responses. A periodic component, resulting from the helical swimming, gradually aligns the helix towards the gradient. When incremental path corrections fail and sperm get off course, a sharp turning manoeuvre puts sperm back on track. Turning results from an ‘off' Ca2+ response signifying a chemoattractant stimulation decrease and, thereby, a drop in cyclic GMP concentration and membrane voltage. These findings highlight the computational sophistication by which sperm sample gradients for deterministic klinotaxis. We provide a conceptual and technical framework for studying microswimmers in 3D chemical landscapes.

Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa

Speract, a peptide from the egg jelly coat of certain sea urchin species, modulates sperm motility through a signaling pathway involving several ionic fluxes leading to pHi and [Ca2+]i increases. [Ca2+]i oscillations in the flagellum regulate its beating pattern modulating sperm swimming. Recent evidence showed the importance of pHi in controlling Ca2+ influx and chemotaxis. However, spatio-temporal characterization of the flagellar pHi increase triggered by speract, and its correlation to that of [Ca2+]i is lacking. Here, we show for the first time in single sea urchin spermatozoa that the speract-induced flagellar pHi increase precedes and is independent of [Ca2+]i increase. Our results support a leading role of pHi in modulating the Ca2+ signals that govern sperm swimming.

Effect of silver nanoparticles on marine organisms belonging to different trophic levels

Silver nanoparticles (Ag-NPs) are increasingly used in a wide range of consumer products and such an extensive use raises questions about their safety and environmental toxicity. We investigated the potential toxicity of Ag-NPs in the marine ecosystem by analyzing the effects on several organisms belonging to different trophic levels. Algae (Dunaliella tertiolecta, Skeletonema costatum), cnidaria (Aurelia aurita jellyfish), crustaceans (Amphibalanus amphitrite and Artemia salina) and echinoderms (Paracentrotus lividus) were exposed to Ag-NPs and different end-points were evaluated: algal growth, ephyra jellyfish immobilization and frequency of pulsations, crustaceans mortality and swimming behavior, and sea urchin sperm motility. Results showed that all the end-points were able to underline a dose-dependent effect. Jellyfish were the most sensitive species, followed by barnacles, sea urchins, green algae, diatoms and brine shrimps. In conclusion, Ag-NPs exposure can influence different trophic levels within the marine ecosystem.

Analysis of Hyperstable Ciliary Microtubule Protofilaments

Eukaryotic cilia and flagella are evolutionarily conserved organelles involved in motility, signal transduction and disease. Their axoneme consists of a cylindrical array of 9 doublet microtubules (DMTs). The objective of this study was to analyze a specialized region of DMTs, i.e., “Ribbons” of 3-4 protofilaments that are stable after extraction with Sarkosyl detergent. Sea urchin sperm flagellar axonemes were analyzed for the distribution and stoichiometry of Ribbon-associated proteins by immuno-biochemistry and proteomics, and their structural arrangement by immuno-electron microscopy and immuno-/cryo-electron tomography. The proteins associated with Ribbons in a reproducible ratio include: acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the ciliary tektins, and >95% of two Ca2+-binding proteins, Rib74 and Rib85.5, whose human homologues are associated with juvenile myoclonic epilepsy. Ribbons can be further fractionated into filaments composed exclusively of tektins. Each DMT contains only one Ribbon, corresponding to protofilaments A11-12-13-1 of the ciliary A-tubule, with a prominent electron density on the side of the Ribbon facing the A-tubule lumen. Each Ribbon contains a single, 5-nm-diameter, hyperstable filament of equimolar tektins A, B and C. Diverse studies indicate that tektins interact with dynein and the nexin-dynein regulatory complex. Ribbon proteins are proposed to function in determining 3-dimensional ciliary architecture, stabilizing ciliary DMTs, and regulating ciliary motility.

Sulfated glycans in sea urchin fertilization.

Fertilization is a controlled cell-cell interaction event that ultimately leads to the union of the gametes involved in reproduction. Fertilization is characterized by three major steps: (i) sperm binding to the extracellular matrix that coats the egg, inducing thereby the acrosome reaction; (ii) penetration of the acrosome-reacted sperm through the egg coat until its contact with the egg plasma membrane; and (iii) adhesion and fusion of the cell membranes of both gametes and the interchange of genetic materials. The acrosome reaction in the first step is important because it ensures that fertilization occurs only between gametes of homologous species. This specificity is primarily driven by the structure of egg jelly coat glycans recognized by a lectin-like binding protein (receptor) in the sperm membrane. Sea urchin fertilization is the best model utilizedfor understanding carbohydrate-mediated acrosome reactions. This report aims at describing the biochemical basis of regulatory mechanisms exerted by sea urchin sulfated fucans and galactans of well-defined chemical structures on the egg-sperm recognition process during fertilization of this invertebrate. Flagellasialin, a sulfated polysialic acid-containing glycoprotein found in sea urchin sperm flagella, is another sulfated glycan example also involved in fertilization of the echinoderm.

Larry Cohen—50 ways to DYE your science

Larry Cohen—50 ways to DYE your science

The CatSper channel controls chemosensation in sea urchin sperm.

Sperm guidance is controlled by chemical and physical cues. In many species, Ca(2+) bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca(2+) bursts. The underlying Ca(2+) channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca(2+) channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca(2+) influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca(2+) bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.

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.

Sperm’s sensitive steering machinery

Pichlo et al. describe how sea urchin sperm respond to tiny amounts of chemoattractant. Sea urchin sperm are incredibly sensitive, being able to detect and respond to single molecules of chemoattractant as they navigate toward the egg. The guanylyl cyclase (GC) chemoreceptor localizes to the sperm

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons.

Influence of low-intensity red diode and laser radiation on the locomotor activity of sea urchin sperm.

Influence of low-intensity red diode and laser radiation on the locomotor activity of sea urchin sperm.

Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules: TEKTINS, Ca2+-BINDING PROTEINS, AND STABLE PROTOFILAMENTS

Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems.

Structural mechanism of the dynein power stroke.

Dyneins are large microtubule motor proteins required for mitosis, intracellular transport, and ciliary and flagellar motility1,2. They generate force through a powerstroke mechanism, which is an ATP-consuming cycle of pre- and post-powerstroke conformational changes that cause relative motion between different dynein domains3-5. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (i.e. beating), rapidly frozen, sea urchin sperm flagella, we determined the in situ 3D structures of all domains of both pre- and post-powerstroke dynein, including the previously unresolved linker and stalk of pre-powerstroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-powerstroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide 3D-reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.