Effects of calcium on the longituindal flagellum of Oxyrrhis marina

Summary— 2–4 nm filaments represent a new class of cytoskeletal components. They are found in ciliary and flagellar roots and centrosomes of all eucaryotes. They are also the major components of paraflagellar rods (PFR) in Euglena, trypanosomes and dinoflagellates. Oxyrrhis marina, a marine dinoflagellate, possesses a transverse and a longitudinal flagellum. Only the longitudinal flagellum carries the PFR along the proximal two-thirds of its length. This flagellum is not only capable of the classic flagellar beat but is also able to retract and bend, a property mediated by external calcium. To determine if calcium has a direct role in the bending, experimental conditions were established to permeabilization and reactivation. Our conditions to reactivate the axoneme function (wave propagation) appear similar to those observed in the case of the sea urchin sperm. The results show that in vitro, an increase in calcium concentration induces a conformational change of the longitudinal flagellum in the absence of ATP with a half maximum effect at 0.1 μM. In the presence of ATP, this morphology modification causes a total inhibition of the wave propagation which is replaced by non-propulsive contractions of low amplitude. As these properties are not shared by reactivated sea urchin sperm flagella or the transverse flagellum of O marina devoid of PFR, we propose that PFR are responsible for the bending phenomenon. A calcium shock also induces flagellar excision with a half maximum effect at 0.3 μM, and immunofluorescence results suggest that a centrin-like protein is present in O marina and is responsible for this excision.     

High-speed video observation of swimming behavior and flagellar motility ofProrocentrum minimum (Dinophyceae)

Summary To understand the functions of the longitudinal and transverse flagella of dinoflagellates, the flagellar waveform and frequency of each flagellum were observed by high-speed video-recording. The longitudinal flagellum emerged from the anterior end of the cell and beat with a planar undulating wave whose plane was perpendicular to the valval sutural plane. The transverse flagellum curved around the anterior end of the cell and beat with a helical wave, with different alternating half pitches. The half pitch corresponding to the parts farther from the cellular antero-posterior axis was shorter than that of the parts closer to the axis. This pattern is described by the ratio of the outer-parts half pitch to the pitch of the whole period of the helix and seems to be characteristic of the dinoflagellates' transverse flagellum.Abbreviations p _in half pitch corresponding to the inner parts of the transverse flagellum - p _out half pitch corresponding to the outer parts of the transverse flagellum - P _p pitch of helical swimming trajectory - R _p radius of helical swimming trajectory - _c rotational frequency of the cell     

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology,regulation of sperm motility and interpopulation diversity

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species‐specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl₂, respectively). Sperm maintained a high motility rate over a wide pH range (5·5–10·5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.     

Insights into the mechanism of ADP action on flagellar motility derived from studies on bull sperm

Adenosine diphosphate (ADP) is known to have interesting effects on flagellar motility. Permeabilized and reactivated bull sperm exhibit a marked reduction in beating frequency and a greatly increased beat amplitude in the presence of 1-4 mM ADP. In this study we examined the force production of sperm reactivated with 0.1 mM ATP with and without 1 mM ADP and found that there is little or no resulting change in the stalling force produced by a bull sperm flagella in response to ADP. Because bull sperm bend to a higher curvature after ADP treatment we explored the possibility that ADP-treated sperm flagella are more flexible. We measured the stiffness of 50 μM sodium vanadate treated bull sperm in the presence of 4 mM ADP, but found no change in the passive flagellar stiffness. When we analyzed the torque that develops in ADP-treated sperm at the point of beat reversal we found that the torque developed by the flagellum is significantly increased. Our torque estimates also allow us to calculate the transverse force (t-force) acting on the flagellum at the point of beat direction reversal. We find that the t-force at the switch-point of the beat is increased significantly in the ADP treated condition, averaging 0.7 ± 0.29 nN/μm in 0.1 mM ATP and increasing to 2.9 ± 1.2 nN/μm in 0.1 mM ATP plus 4 mM ADP. This suggests that ADP is exerting its effect on the beat by increasing the tenacity of dynein attachment at the B-subtubule. This could be a direct result of a regulatory effect of ADP on the binding affinity of dynein for the B-subtubule of the outer doublets. This result could also help to explain a number of previous experimental observations, as discussed.     

Motion of the Longitudinal Flagellum in Ceratium tripos (Dinoflagellida): A Retractile Flagellar Motion1

The longitudinal flagellum of Ceratium tripos moves in two dissimilar ways: undulation and retraction. The undulatory wave is planar and has a wavelength of 74.3 ± 9.6 μm and an amplitude of 14.2 ± 2.3 μm in sea water. The beat frequency is 30 Hz at 20°C, pH 8.0. The retractile motion is unique to Ceratium and is triggered by mechanical stimulation on the cell body, especially at the tip of the apical horn. When it retracts, the longitudinal flagellum folds every 4–5 μm along the flagellum. Cinematographic study showed that the flagellum folded from tip to base and was finally installed into the sulcus, a groove on the ventral side of the cell. This motion is completed in sea water within 28 msec. The retracted flagellum then re-extends and restores the undulation within a few seconds. The flagellum unfolds in the proximal portion first, then the distal, and finally the middle portion. Fixation always triggers the retraction. Scanning electron microscopy showed that the flagellum is folded and secondarily twisted in a helix. A new fiber in addition to the flagellar axoneme was found in the retracted flagellum by phase microscopy. This fiber (R-fiber) seems to contract during the retraction to fold the flagellum.     

Sperm traits in farmed and wild Atlantic salmon Salmo salar

Differences in sperm metabolism and morphology between wild and non‐local farmed Atlantic salmon Salmo salar were assessed by measuring metabolic enzyme activities and length of sperm flagella. No differences were observed between wild and farmed S. salar sperm with regards to cell counts or any of the biochemical variables assessed. Flagella of sperm cells were significantly longer in wild than farmed S. salar; however, this did not result in higher energy levels or different fertilization rates.     

A selective effect of Ni2+ on wave initiation in bull sperm flagella

Bull sperm that are extracted with 0.1% Triton X-100 and restored to motility with Mg2+-ATP lose coordination and stop swimming in the presence of 0.5 mM NiSO4. Although spontaneous coordination of flagellar waves is lost after exposure to Ni2+, other functions of the flagellum remain intact. The capacity for wave propagation along the flagellum is maintained together with the capacity for microtubular sliding. Wave motility can be restored to Ni2+-inhibited sperm by inducing a permanent bend onto the flagellum by micromanipulation. In the absence of such intervention, the loss of wave coordination is complete and irreversible. Ni2+-inhibited demembranated cells that are kept active by maintaining a bend in the flagellum exhibit a normal beat frequency. Both intact and demembranated sperm can retain spontaneous wave production at considerably slower rates of motion than Ni2+-inhibited cells. Short segments from the distal tip of the flagellum contain only the 9 + 2 microtubular axoneme. These short segments are able to propagate imposed bends even in the presence of Ni2+. In addition to wave propagation Ni2+-treated sperm can be shown to exhibit a normal sliding tubule phenomenon by direct assay. Although Ni2+-treated cells have a functional sliding tubule mechanism, and consequently the axoneme can propagate bends, it appears that these retained functions are not sufficient to cause spontaneous bend initiation. Our findings show that bend initiation is inhibited by Ni2+, and therefore is an independent process separate from the sliding tubule mechanism responsible for wave propagation.     

ULTRASTRUCTURE OF SWARMERS IN THE LAMINARIALES (PHAEOPHYCEAE). II. SPERM1

The ultrastructure of sperm from 13 species in 11 genera of Laminariales collected in the northeast Pacific Ocean is unique in the brown algae. The sperm are elongate, and possess a nucleus, several mitochondria and two or three chloroplasts, but no eyespot. The anterior flagellum bears mastigonemes on the proximal half of its length; a distal “whiplash” portion lacks mastigonemes and is an extension of only the two central singlet microtubules of the axoneme. A peculiar feature of these sperm is the posterior flagellum, which is longer than the anterior flagellum and tapers distally as the doublet microtubules become singlets and decrease in number. This feature contrasts with the laminarialean zoospore, which possesses a short posterior flagellum with the usual “9 + 2” axoneme. The structure of these sperm differs from that reported for Chorda, the sperm of which resembles a primitive brown algal zoospore. The facts support the concept that Chorda is the most primitive member of the Laminariales.     

Reversible intracellular ATP changes in intact rat spermatozoa and effects on flagellar sperm movement.

The initiation of motility and modification of energy metabolism of rat caudal epididymal spermatozoa can be induced by dilution in a saline medium. We have investigated in these cells the relationships between the energy reserve (sperm ATP content measured by bioluminescence) and flagellar movement (high speed videomicrography, 200 frames/sec). A steady state was observed in sperm ATP content, progressive velocity (Vp) and flagellar beat frequency (F) with sperm dilution in a medium with glucose, lactate, pyruvate and acetate substrates after 30 minutes of incubation. Without these substrates, changes in metabolic pathways occurred immediately and initially disturbed the relationship between ATP levels and F, suggesting differences in motility initiation when energy is from an endogenous origin via mitochondrial oxidative phosphorylation. This "energy crisis" was reversed by the addition of substrates to the medium. The three-dimensional flagellar movement observed in the presence of substrates quickly became two-dimensional in their absence. The flagellar beat envelope became more splayed, the mean amplitude of lateral head displacement increased and F decreased. The resulting high flagellar beat efficiency can be compared to that observed during hyperactivation which is a physiological event related to a fall in intracellular ATP level. In both media, the displacement of the flagellum in relation to the wave axis varied sinusoidally. The sine period increased with time when the spermatozoa were incubated in the medium without substrates. These results suggest a gradual slowing-down of the velocity of wave formation in the proximal part of the flagellum.     

Ultrastructural observations on membrane changes associated with cryopreserved spermatozoa of two polychaete species and subsequent mobility induced by quinacine

Summary

A wide range of cryoprotectants and cooling rates have been tested on the spermatozoa of two polychaete species Arenicola marina and Nereis virens. The techniques were more successful with N. virens; with this species active sperm were recovered after immersion in liquid nitrogen following a variety of cooling protocols. Structural damage to the sperm of both species, however, was observed following freezing, and this was more severe in the case of A. marina. Structural damage of the flagellum plasma membrane was most commonly observed. Biologically important changes in the sperm can be induced by the freezing procedures; for example acrosome eversion may be induced in N. virens sperm. Dissociation of the sperm of A. marina can be induced after freezing by the sperm activator quinacrine, but these sperm do not acquire forward motility probably due to flagellum damage.     

Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development

Background  

The sperm annulus is a septin-based fibrous ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Although ultrastructural abnormalities and functional importance of the annulus have been addressed in Sept4-null mutant mice and a subset of human patients with asthenospermia syndrome, little is known about how the structure is assembled and positioned to the midpiece-principal piece junction during mammalian sperm flagellum development.     

THE RELEASE MECHANISM AND LOCOMOTOR BEHAVIOR OF EQUISETUM SPERM

When antheridia of gametophytes of Equisetum hyemale L. are placed in water, only spermatid cells are released. These spermatid cells have from 6–12 fibrils radiating from 2–4 loci on the cell wall. One sperm is released from each spermatid cell. With high speed cinemicrography, it can be shown that sperm flagella beat three dimensionally with a continuous, traveling helical wave. A flagellar beat cycle is completed every 0.03 sec. In water the sperm swim rapidly at a rate of 300 μ per sec and traverse a helical path of long wavelength and shallow amplitude. Calcium is essential for normal locomotor behavior. When calcium is chelated by ethylenediaminetetraacetate (EDTA), the sperm spin in place. All other monovalent and divalent ionic solutions tested cause the sperm to swim abnormally. Monovalent ionic solutions prevent the drastic change in sperm mobility patterns resulting from chelation by EDTA.     

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.     

Trypanosomes and mammalian sperm: one of a kind?

Flagellar-mediated motility is an indispensable function for cell types as evolutionarily distant as mammalian sperm and kinetoplastid parasites, a large group of flagellated protozoa that includes several important human pathogens. Despite the obvious importance of flagellar motility, little is known about the signalling processes that direct the frequency and wave shape of the flagellar beat, or those that provide the motile cell with the necessary environmental cues that enable it to aim its movement. Similarly, the energetics of the flagellar beat and the problem of a sufficient ATP supply along the entire length of the beating flagellum remain to be explored. Recent proteome projects studying the flagella of mammalian sperm and kinetoplastid parasites have provided important information and have indicated a surprising degree of similarities between the flagella of these two cell types.     

Dealing with several flagella in the same cell

Flagella are sophisticated organelles found in many eukaryotic microbes where they perform functions related to motility, signal detection, or cell morphogenesis. In many cases, several flagella are present per cell, and these can have a different composition, length, age, or function, raising the question of how this is managed. When the flagella are equivalent and constructed simultaneously such as in Chlamydomonas or Naegleria, we propose an equal access model where molecular components have free access to each organelle. By contrast, Trypanosoma and Leishmania contain temporally distinct organelles and elongate a new flagellum whilst maintaining the existing one. The equal access model could function providing that the mature flagellum is “locked” so that it can no longer be elongated or shortened. Alternatively, access of flagellar components could be restricted at the level of the basal body, the transition zone, or the loading on intraflagellar transport trains. In organisms that contains flagella of different age and composition such as Giardia, a temporal dimension is necessary, with the production of protein components of flagella spreading over one or more cell cycles. In the future, deciphering the molecular mechanisms involved in these processes should reveal new insights in flagellum assembly and function.     

Morphologically Specialized Sperm from the Ovary of Isometra vivipara (Echinodermata — Crinoidea)

The morphology of the sperm of Isometra vivipara is markedly different from the spherical-headed sperm typical of crinoids in general. The sperm head of Isometra has a flattened, oval shape. In addition to a nucleus, the head includes a lateral acrosome on one flat surface and a lateral mitochondrion on the opposite flat surface. From the latter surface of the sperm head, a tail flagellum arises adjacent to the mitochondrion. The discussion considers the relationships between reproductive habits and sperm morphology in Isometra and other crinoids.     

Sperm motility and calcium transport: a neurochemically controlled process

Elucidation of the functional significance of the presence of acetylcholinesterase in spermatozoa was deferred until considerably after the initial observations that eserine increased flagellar beat frequency in $\text{Mytilus}$ sperm (54). Discovered about forty years ago during surveys of seminal plasma constituents (2), the enzyme was later found to be associated with the particulate fraction (4). Even though characterized as a “true” acetylcholinesterase in the sperm flagellum (3), interpretations of the role of acetylcholine as a factor in motile processes remained speculative until a wide variety of cholinergic agents was found to affect sperm cell performance (32). Those agents which inhibit acetylcholine synthesis or block cholinergic receptors generally elicit a monophasic, inhibitory, dose-dependent response on sea urchin sperm swimming speed. These include hemicholinium, curare, α-bungarotoxin and decamethonium. Cholinomimetic substances and anticholinesterases (acetylcholine and nicotine; eserine, diisopropylfluorophosphate and neostigmine) act biphasically to increase the sperm swim rate at concentrations of 1 micromolar or less while higher concentrations slow or stop the forward motion of the sperm cells. Procaine (7), which competes with Ca²⁺ for cell surface binding sites, initially stimulates then completely inhinits sperm cell progression. Other substances which interfere with the cells′ access to Ca²⁺ or with the transport of Ca²⁺ into and within the cell adversely affect spermatozoan function. A conceptual model has been proposed, a) to relate the uptake of Ca²⁺ to cholinergic regulation of ionophoric channels through the sperm cell plasma membrane and b) for this Ca²⁺ to trigger release of sequestered calcium for coupling of excitation to flagellar wave propagation.     

The sperm structure of Cryptocercus punctulatus Scudder (Blattodea) and sperm evolution in Dictyoptera

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three‐layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provided with accessory tubules with 16 protofilaments and intertubular material. These sperm characters are shared with several other polyneopterans. The sperm of C. punctulatus is very similar to what is found in Periplaneta americana and species of other groups of roaches, including the sperm of Loboptera decipiens described here for the first time. The general sperm organization here described can be assumed for the groundplan of Insecta and Pterygota. The following evolutionary path can be suggested: after the split between Cryptocercidae and the common ancestor of Isoptera, the typical pattern of sperm formation was altered very distinctly, resulting in a duplication or multiplication (Mastotermitidae) of the centrioles. Mastotermes has maintained a certain sperm motility, but with a very unusual apparatus of multiple flagella with a 9+0 axoneme pattern. After the split into Mastotermitidae and the remaining Isoptera, sperm motility was completely abandoned, and different modifications of sperm components occurred, and even the loss of the sperm flagellum. J. Morphol. 276:361–369, 2015. © 2014 Wiley Periodicals, Inc.     

Relationship between semen characteristics and body size in Barbus barbus L. (Teleostei: Cyprinidae) and effects of ions and osmolality on sperm motility

The objectives of the present study were to determine the relationships among length and weight of males, sperm volume, spermatozoa concentration, total number of spermatozoa, ionic contents and osmolality of seminal plasma in Barbus barbus. The effect of osmolality on sperm motility parameters after activation in NaCl, KCl, or sucrose solutions was also examined. There were significant correlations between spermatozoa concentration – length (R = + 0.7) and – weight (R = + 0.8) of males. No significant correlations were observed between the total number of spermatozoa, sperm volume, and length and weight of males. Seminal plasma osmolality was higher when the total number of spermatozoa (R = + 0.6) and sperm volume (R = + 0.6) were higher. Sperm motility and velocity was positively correlated with osmolality (R = + 0.5). The correlation between sperm motility and K⁺ was negative (R = 0.5), but positively correlated with Ca²⁺ (R = 0.8), Na⁺ (R = 0.8), and Cl⁻ (R = 0.8). There was a rapid decrease (P < 0.05) in sperm motility parameters after sperm activation. Just after sperm activation, beating waves propagated along the full length of flagella. At latter stages post sperm activation, the waves appeared only in proximal part of the flagellum. The highest spermatozoa velocity and percentage of motility were observed at 215–235 mOsmol kg^− 1 in NaCl, KCl or sucrose. The tip of the flagellum became curled into a loop shape which shortened the flagellum after activation of sperm in distilled water. B. barbus sperm is very similar to that of other cyprinids in terms of ionic contents and osmolality of the seminal plasma, mechanism of sperm activation and behavior and motility of sperm during swimming period.