Motile Male Gametes of the Araphid Diatom Tabularia fasciculata Search Randomly for Mates

Sexuality in the marine araphid diatom Tabularia involves an unusual type of gamete, not only among diatoms but possibly in all of nature. The non-flagellated male gamete is free and vigorously motile, propelled by pseudopodia. However, the cues (if any) in their search for compatible female gametes and the general search patterns to locate them are unknown. We tracked and compared male gamete movements in the presence and absence of receptive female gametes. Path linearity of male movement was not affected by presence of female gametes. Male gametes did not move towards female gametes regardless of their proximity to each other, suggesting that the detection range for a compatible mate is very small compared to known algal examples (mostly spermatozoids) and that mate recognition requires (near) contact with a female gamete. We therefore investigated how male gametes move to bring insight into their search strategy and found that it was consistent with the predictions of a random-walk model with changes in direction coming from an even distribution. We further investigated the type of random walk by determining the best-fit distribution on the tail of the move length distribution and found it to be consistent with a truncated power law distribution with an exponent of 2.34. Although consistent with a Lévy walk search pattern, the range of move lengths in the tail was too narrow for Lévy properties to emerge and so would be best described as Brownian motion. This is somewhat surprising because female gametes were often outnumbered by male gametes, thus contrary to the assumption that a Brownian search mode may be most optimal with an abundant target resource. This is also the first mathematically analysed search pattern of a non-flagellated protistan gamete, supporting the notion that principles of Brownian motion have wide application in biology.     

A role for sperm surface protein disulfide isomerase activity in gamete fusion: evidence for the participation of ERp57

In mammals, sperm-egg interaction is based on molecular events either unique to gametes or also present in somatic cells. In gamete fusion, it is unknown which features are gamete specific and which are shared with other systems. Conformational changes mediated by thiol-disulfide exchange are involved in the activation of some virus membrane fusion proteins. Here we asked whether that mechanism is also operative in sperm-egg fusion. Different inhibitors of protein disulfide isomerase (PDI) activity were able to inhibit sperm-egg fusion in vitro. While pretreatment of oocytes had no effect, pretreatment of sperm reduced their fusion ability. Some members of the PDI family were detected on the sperm head, and use of specific antibodies and substrates suggested that the oxidoreductase ERp57 has a role in gamete fusion. The results support the idea that thiol-disulfide exchange is a mechanism that may act in gamete fusion to produce conformational changes in fusion-active proteins.     

Photographs of Fertilization in the Smaller Species of Trichonympha

SYNOPSIS. The photographs illustrate male and female gametes before fertilization, several progressive stages in the entrance of the male gamete into the cytoplasm of the female, cytoplasmic fusion of gametes, loss of extranuclear organelles of male gamete, retention of extranuclear organelles of female gamete, movement of pronucleus of male gamete to that of female, progressive stages in fusion of pronuclei, and the formation of the zygote which possesses the extranuclear organelles of the female gamete. Some abortive attempts at fertilization, resulting from failure of gametes to differentiate, are shown.     

Population dynamics of sperm and pollen killers

Summary A model of segregation distortion is assumed in which the action of the distorter when heterozygous is to render dysfunctional those gametes that carry its allele. Two gamete killers when homozygous are assumed to distort each other. Individuals that carry the gamete killer suffer a reduction in the number of functional gametes they produce, but this deleterious effect is counterbalanced by the segregation ratio advantage of the distorter. The dynamics of such a system are analyzed in terms of a generalized fecundity function, which is defined as a function which assigns to any individual his relative fecundity in terms of the fraction of functional gametes he produces. Three general classes of fecundity functions are considered: (a) proportionality, in which the relative fecundity of an individual is proportional to the fraction of functional gametes he produces, (b) compensation, in which the relative fecundity of an individual is always greater than the fraction of functional gametes he produces, and (c) mass action, in which the relative fecundity of an individual is less than or greater than the fraction of functional gametes he produces according to whether the fraction of functional gametes is less than or greater than some threshold. In case (a) all gamete killers are always at neutral equilibria and gene frequency changes at the locus are governed by random drift. In case (b) all gamete killers will be fixed if the fecundity function is such that its second derivative is negative, whenever its argument is greater than one-half. And in case (c) some gamete killers will converge to stable equilibria, others will be fixed. If a gamete killer is homozygous lethal it will almost always converge to a stable equilibrium.This work was made possible by grant number GB-18786 from the National Science Foundation.     

Mechanism of male gamete motility in araphid pennate diatoms from the genus Tabularia (Bacillariophyta)

During sexual reproduction, araphid pennate diatoms of the genus Tabularia (Kützing) D. M. Williams and Round released male gametes directly into the medium, sometimes at a considerable distance from the female gametes. This raised the question of how male gametes, suspended in water, manage to reach female ones, given that no locomotive organelles have been described in gametes of pennate diatoms. Optical microscopic investigation revealed cytoplasmic projections produced by male gametes of Tabularia tabulata (C. A. Agardh) Snoeijs and T. fasciculata (C. A. Agardh) D. M. Williams and Round. Morphology and behavior of these projections is consistent with pseudopodia, however, which specific type of pseudopodia they may be, remains inconclusive. The growth and retraction of the pseudopodia coincided with gamete motility and so we postulate that it explains the otherwise apparent random movement of male gametes. Spinning, shuffling and chaotic patterns of motility were documented. In theory, gamete mobility increases the probability of gamete encounter thus enhancing the probability of syngamy. This is the first known case where cytoplasmic projections have been described in diatom gametes, and possibly in mature gametes in general.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

Blue- and green-light signals for gamete release in the brown alga, Silvetia compressa

The intertidal brown alga Silvetia compressa releases gametes from receptacles (the reproductive tissue) rapidly upon a dark transfer (following a photosynthesis-dependent period in the light, termed potentiation). In this study, the wavelength-dependence of this process was investigated. During the potentiation period in white light (WL), gametes are not released. However, gametes were released during potentiation in blue light (BL), or in low red light/blue light (RL/BL) ratios, but not in RL alone, high RL/BL ratios, or in broadband blue-green light (B-GL) (presence of BL, but absence of RL). RL was as effective as WL for potentiation, i.e., both lead to gamete release following transfer to darkness. Rates of linear photosynthetic electron transport were similar in RL and BL. Gamete release in BL was inhibited by equal amounts of additional narrow-waveband light between the green and red regions of the spectrum, with light-induced gamete release restricted between <491 nm and 509 nm. Very little light-induced gamete release occurred between 530 nm and 650 nm. It is proposed that a BL-responsive photoreceptor is responsible for light-induced gamete release. Transfer of WL-potentiated receptacles to GL near 530 nm resulted in significant de-potentiation and reduced gamete release during a subsequent dark transfer. This effect was not seen at 509 nm or 560 nm and revealed the presence of a second photoreceptor system repressing or counteracting potentiation in the light. We propose that the restriction of gamete release to periods when irradiance is blue-shifted may constitute a depth-sensing mechanism for this intertidal alga, allowing controlled release of gametes at high tide and/or less turbid periods, thus minimizing gamete dilution, and promoting fertilization success.     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Analysis of gamete and zygote motility in Allomyces

To study the mechanisms of chemotaxis in eukaryotes, the motility patterns of the gametes and zygotes and the chemotactic responses of the male gametes of the lower eukaryote Allomyces macrogynus were examined. Dark-field microscopy of the male gametes showed a smooth swimming pattern interrupted by very brief ‘jerks’ of the cell body that caused a change in swimming direction. Female gametes had a slower swimming velocity than the males and underwent more jerks or turns which accounted for their sluggish motility. The zygotes swam with the fastest velocity and were observed to have a helical swimming pattern involving a continuous turning of the cell body, a behavior absent from the gametes. Introduction of female gametes that produce the chemoattractant sirenin brought about an immediate change in the behavior of the male gametes. They moved in spirals (or helices) towards the source of the chemoattractant (the female gametes), undergoing only a few jerks to reorient the male cells. When very near the female cells, or in high concentrations of added sirenin, many very short motility tracks were observed that finally resulted in contact between the two gamete types. The results indicate that the poor swimming ability of the female gametes facilitates gamete contact, resulting in as many as 30–40 male gametes clustered on a single female cell. Further, male gamete orientation to the sirenin gradient is caused by the chemoattractant suppressing the jerk motion.     

On the asymmetry of sex: Evolution of mating types in isogamous populations

This paper is an analysis using population genetic models of the problem of the evolution of two mating types from an original situation in which all gametes are alike functionally (i.e. each gamete can fuse with any other gamete). It is shown that inbreeding depression due to fusion of identical gametes from the same clone is unlikely to play a significant role in this evolution. Evolution towards unipolarity in gamete recognition or adhesion (implying the existence of two mating types) requires a more than twofold disadvantage for bipolarity. However, the evolution of mating types is greatly facilitated if a pheromonal gamete attraction mechanism is already present. The models finally predict the evolution of a mating type “super gene” determining the various functions associated with mating.     

Regulated targeting of a protein kinase into an intact flagellum. An aurora/Ipl1p-like protein kinase translocates from the cell body into the flagella during gamete activation in chlamydomonas

In the green alga Chlamydomonas reinhardtii flagellar adhesion between gametes of opposite mating types leads to rapid cellular changes, events collectively termed gamete activation, that prepare the gametes for cell-cell fusion. As is true for gametes of most organisms, the cellular and molecular mechanisms that underlie gamete activation are poorly understood. Here we report on the regulated movement of a newly identified protein kinase, Chlamydomonas aurora/Ipl1p-like protein kinase (CALK), from the cell body to the flagella during gamete activation. CALK encodes a protein of 769 amino acids and is the newest member of the aurora/Ipl1p protein kinase family. Immunoblotting with an anti-CALK antibody showed that CALK was present as a 78/80-kDa doublet in vegetative cells and unactivated gametes of both mating types and was localized primarily in cell bodies. In cells undergoing fertilization, the 78-kDa CALK was rapidly targeted to the flagella, and within 5 min after mixing gametes of opposite mating types, the level of CALK in the flagella began to approach levels normally found in the cell body. Protein synthesis was not required for targeting, indicating that the translocated CALK and the cellular molecules required for its movement are present in unactivated gametes. CALK was also translocated to the flagella during flagellar adhesion of nonfusing mutant gametes, demonstrating that cell fusion was not required for movement. Finally, the requirement for flagellar adhesion could be bypassed; incubation of cells of a single mating type in dibutyryl cAMP led to CALK translocation to flagella in gametes but not vegetative cells. These experiments document a new event in gamete activation in Chlamydomonas and reveal the existence of a mechanism for regulated translocation of molecules into an intact flagellum.     

Modulation of the oviductal environment by gametes

The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.     

Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

The iso1 gene of Chlamydomonas is involved in sex determination.

Sexual differentiation in the heterothallic alga Chlamydomonas reinhardtii is controlled by two mating-type loci, mt+ and mt-, which behave as a pair of alleles but contain different DNA sequences. A mutation in the mt minus-linked imp11 gene has been shown previously to convert a minus gamete into a pseudo-plus gamete that expresses all the plus gametic traits except the few encoded by the mt+ locus. Here we describe the iso1 mutation which is unlinked to the mt- locus but is expressed only in minus gametes (sex-limited expression). A population of minus gametes carrying the iso1 mutation behaves as a mixture of minus and pseudo-plus gametes: the gametes isoagglutinate but they do not fuse to form zygotes. Further analysis reveals that individual gametes express either plus or minus traits: a given cell displays one type of agglutinin (flagellar glycoprotein used for sexual adhesion) and one type of mating structure. The iso1 mutation identifies a gene unlinked to the mating-type locus that is involved in sex determination and the repression of plus-specific genes.     

The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes

The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mt+ and mt- gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a approximately 95-kDa protein present on the external surface of both unactivated and activated mt+ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mt+ gametes at the site of cell-cell fusion and essential for an early step in the fusion process.     

Production of anisogametes and gamete motility dimorphism in Monostroma angicava

 The reproductive strategy of a marine alga with a heteromorphic biphasic life cycle was studied by analyzing various sexual reproductive characters in light of the evolution of anisogamy. Gametophytes of Monostroma angicava were dioecious and their gametes were slightly anisogamous. Volume of gametangium, density of gametangia and area of mature gametangial parts on each gametophyte did not differ from male to female. Therefore, the reproductive biomass investment for gamete production was considered to be the same for each sex. Anisogamy in this alga appeared to be derived from the difference in the number of cell divisions during gametogenesis, because the majority of male gametangia each produced 64 (2⁶) gametes and the female produced 32 (2⁵) gametes. This corresponded with measurements of cell size in male and female gametes. Further, the sex ratio was 1:1 for sexually mature plants sampled at Charatsunai. Therefore, it was suggested that in the field twice as many male gametes are released as female gametes. Liberated gametes of both sexes showed positive phototaxis. The swimming velocity of freshly liberated male gametes was a little higher than that of female gametes. Male gametes had the potential to swim for ca. 72 h and female gametes for ca. 84 h. The difference in gamete motility between the two sexes seemed to be related to cell size. Planozygotes were negatively phototactic and swam more rapidly than gametes of either sex. Received: 5 March 1997 / Revision accepted: 18 July 1997     

Research note: Induction of gamete discharge by hypertonic treatment in the green alga Bryopsis plumosa (Caulerpales,Chlorophyta)

Brief treatment with hypertonic solutions induced gamete discharge from gametangia in the coenocytic green alga Bryopsis plumosa (Hudson) C. Agardh. It is known that gamete discharge in this alga is triggered by a change from darkness to light. However, in this study mature gametangia, incubated for 30–120 s in artificial seawater (ASW) with an additional 0.4–0.6 M NaCl and then transferred into pure ASW, discharged gametes in darkness. The treatment did not affect the motility of the gametes. Addition of sucrose (1.0–1.2 M) to ASW also induced gamete discharge in darkness. Similar results were obtained by adding KCl (0.4 M) or mannitol (1.2 M) to ASW. Continuous incubation of gametangia in such hypertonic solutions also induced gamete discharge but led to a delay and a reduction in the rate of gamete discharge, and a loss of gamete motility. In gametangia treated with the hypertonic solutions, as well as in those exposed to light, shortening of the gametangial length was observed before gamete discharge.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Kinesin-II is required for flagellar sensory transduction during fertilization in Chlamydomonas

The assembly and maintenance of eucaryotic flagella and cilia depend on the microtubule motor, kinesin-II. This plus end-directed motor carries intraflagellar transport particles from the base to the tip of the organelle, where structural components of the axoneme are assembled. Here we test the idea that kinesin-II also is essential for signal transduction. When mating-type plus (mt+) and mating-type minus (mt-) gametes of the unicellular green alga Chlamydomonas are mixed together, binding interactions between mt+ and mt- flagellar adhesion molecules, the agglutinins, initiate a signaling pathway that leads to increases in intracellular cAMP, gamete activation, and zygote formation. A critical question in Chlamydomonas fertilization has been how agglutinin interactions are coupled to increases in intracellular cAMP. Recently, fla10 gametes with a temperature-sensitive defect in FLA10 kinesin-II were found to not form zygotes at the restrictive temperature (32 degrees C). We found that, although the rates and extents of flagellar adhesion in fla10 gametes at 32 degrees C are indistinguishable from wild-type gametes, the cells do not undergo gamete activation. On the other hand, fla10 gametes at 32 degrees C regulated agglutinin location and underwent gamete fusion when the cells were incubated in dibutyryl cAMP, indicating that their capacity to respond to the cAMP signal was intact. We show that the cellular defect in the fla10 gametes at 32 degrees C is a failure to undergo increases in cAMP during flagella adhesion. Thus, in addition to being essential for assembly and maintenance of the structural components of flagella, kinesin-II/intraflagellar transport plays a role in sensory transduction in these organelles.