Spermatogenesis and auxospore structure in the multipolar centric diatom Hydrosera

Spermatogenesis and auxospore development were studied in the freshwater centric diatom Hydrosera triquetra. Spermatogenesis was unusual, lacking depauperating cell divisions within the spermatogonangium. Instead, a series of mitoses occurred within an undivided cell to produce a multinucleate plasmodium with peripheral nuclei, which then underwent meiosis. 32 or 64 sperm budded off from the plasmodium leaving a large residual cell containing all the chloroplasts. Similar development apparently occurs in Pleurosira, Aulacodiscus, and Guinardia, these being so distantly related that independent evolution of plasmodial spermatogenesis seems likely. After presumed fertilization, the Hydrosera egg cell expanded distally to form a triangular end part. However, unlike in other triangular diatoms (Lithodesmium, Triceratium), the development of triradiate symmetry was not controlled by the “canonical” method of a perizonium that constrains expansion to small terminal areas of the auxospore wall. Instead, the auxospore wall lacked a perizonium and possessed only scales and a dense mat of thin, apparently entangled strips of imperforate silica. No such structures have been reported from any other centric diatoms, the closest analogs being instead the incunabular strips of some raphid diatoms (Nitzschia and Pinnularia). Whether these silica structures are formed by the normal method (intracellular deposition within a silica deposition vesicle) is unknown. As well as being more rounded than vegetative cells, the initial cell is aberrant in its structure, since it has a less polarized distribution of the “triptych” pores characteristic of the species.     

AUXOSPORE FORMATION AND THE MORPHOLOGY OF THE INITIAL CELL OF THE MARINE ARAPHID DIATOM GEPHYRIA MEDIA (BACILLARIOPHYCEAE)1

Recent studies have led to a rapid increase in knowledge of auxospore formation in diatoms. However, these studies have been limited to centric and raphid pennate diatoms, and there is still very little information for the araphid pennate diatoms. Using LM and SEM, we studied the development of the auxospore and the initial cell of the marine epiphytic diatom Gephyria media Arnott. Auxospores were bipolar and curved in side view, as in many other pennate diatoms. SEM revealed many transverse perizonial bands, all of which were incomplete rings. There was an elongate, sprawling, silicified structure beneath the ventral suture of the transverse perizonial bands. This structure is presumably equivalent to the longitudinal perizonial band in other pennate diatoms, although we could not determine the homologous relationship between the two features. Scales were found both in the inner wall of the perizonium and around the primary perizonial bands. The presence or absence of scales may be of phylogenetic significance in diatoms, only during the final stages of auxospore formation because scales are found in early spherical stages. The distinctive finger‐like structures observed throughout all stage of G. media have not been observed before in the other diatom taxa.     

Successive observations on the fertilization of a centric diatomMelosira moniliformis var.octagona

The details of fertilization in the centric diatom,Melosira moniliformis var.octagona, were examined by light microscopy with a video system. The sperm penetrated into the egg cell through a temporary opening of the oogonium. The flagellum of the sperm was brought into the egg cell and it remained active for a few minutes. Three observations on the behavior of the sperm and egg cell during fertilization are shown.     

SCALY INCUNABULA,AUXOSPORE DEVELOPMENT,AND GIRDLE POLYMORPHISM IN SELLAPHORA MARVANII SP. NOV. (BACILLARIOPHYCEAE)1

Uniparental auxosporulation was observed in a monoclonal culture of a Sellaphora clone isolated from the epipelon of a fishpond in the Czech Republic. The cox1 sequence for the clone confirmed that it belonged to the Sellaphora pupula–bacillum species complex but showed significant differences from all previously characterized Sellaphora species, and it is therefore described as S. marvanii sp. nov. Protoplast, valve, and girdle structure resembled those of other Sellaphora species, but a novel finding for all diatoms was a change in girdle structure during the life cycle: the most advalvar girdle band (valvocopula) bore a single line of pores in enlarged postauxospore cells but was entirely plain in small cells and gametangia. The young auxospores were covered by incunabula containing large, delicate, ± circular scales, resembling those of centric diatom auxospores; similar scales have been reported in a few other raphid diatoms (Pseudo‐nitzschia multiseries, Diploneis sp.) but contrast with the strip incunabula of some Nitzschia and Pinnularia and the helmet‐like caps of Neidium. The scales persisted during auxospore expansion, mostly as two caps over the auxospore poles. The transverse perizonium comprised a very wide, closed primary band, flanked by numerous secondary bands whose open ends were strongly incurved toward the center. Initial valves were differentiated from their immediate descendants by the very strong external demarcation of the raphe sternum, irregular shape, and curved transapical profile.     

Sperm incorporation in Xenopus laevis: characterisation of morphological events and the role of microfilaments

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.     

Effects of sperm concentration and egg number on fertilization efficiency with channel catfish (Ictalurus punctatus) eggs and blue catfish (I. furcatus) spermatozoa

The mean sperm concentration of 10 blue catfish (Ictalurus furcatus ) was 1.03 x 10(10) per gram of testis. Testis weighed 3.9 and 17.2 g, with a mean of 6.6 g per fish. Fertilization rate of channel catfish (Ictalurus punctatus ) eggs fertilized with 5.00 x 10(4) to 1.20 x 10(7) blue catfish spermatozoa per egg was 17 to 87%, with an overall mean of 65%. Sperm concentrations of 5.0 x 10(4)/egg exhibited a lower, 16.6% (P < 0.05) fertilization rate than higher sperm concentrations (1.25 x 10(5) to 1.20 x 10(8)/egg). Batches of 450, 2,000, 5,000, 8,000 and 11,000 eggs were similarly fertilized with various sperm concentrations. Mean fertility rate ranged from 25 to 67%, with an overall mean of 53%. The largest egg mass produced the lowest (P < 0.05) fertilization rate. A combination of 450 eggs per batch and 5.0 x 10(5) to 1.20 x (8) sperm per egg produced the highest rate of fertilization (67 to 87%).     

ELECTRON MICROSCOPIC OBSERVATIONS OF GUINEA PIG SPERMATOZOA PENETRATING EGGS IN VITRO*

Guinea pig ovarian oocytes matured in vitro were inseminated in vitro with capacitated, acrosome-reacted spermatozoa and sperm penetration through the zona pellucida and into the egg cytoplasm were examined. Sperm heads passing through the zona pellucida had already lost all their acrosomal elements except for the inner acrosomal membrane and the equatorial segment. It was often observed that the texture of the zona material around the sperm head was distorted, giving the impression that the zona pellucida was parted, at least partially, by a shearing force produced by the sperm head advancing through the zona. When eggs were freed from their zonae pellucidae and inseminated, the acrosome-reacted spermatozoa immediately bound to the egg surfaces and began to fuse with the eggs; whereas the spermatozoa with intact acrosomes failed to do so. Fusion began between the egg plasma membrane and the sperm plasma membrane at the central region of the sperm head. The anterior half of the sperm head was engulfed by the egg in a phagocytic fashion, while its posterior half was incorporated into the egg by a fussion between egg and sperm plasma membranes. Incorporation of the sperm tail into the egg was achieved by fusion between the sperm and egg plasma membranes.     

Mechanism of univalent antisperm antibody inhibition of fertilization in the sea urchin, Arbacia punctulata

Univalent antisperm antibodies (IFab) markedly inhibited the fertilizing capacity of sperm when tested on intact, dejellied, and "demembranated" Arbacia punctulata eggs. Sperm motility and egg jelly penetration were not affected by IFab. Antifertilizin was excluded as the essential sperm antigen involved in the fertilization-inhibiting action. Sperm pretreated with IFab did not bind to the surfaces of either dejellied or demembranated eggs, whereas control globulin (CFab) and seawater-pretreated sperm bound to such eggs in high numbers. Electron microscopy showed that IFab-treated sperm failed to undergo the acrosome reaction. This excluded "bindin" as the essential antigen. Inhibition of fertilization by IFab was reversed or bypassed by artificial induction of the acrosome reaction with ionophore A23187. It is concluded that univalent antisperm antibody treatment inhibits the fertilizing capacity of sperm by preventing a sperm-egg interaction that results in the acrosome reaction; consequently, attachment of the sperm to the egg is prevented.     

Sperm chemotaxis in marine species is optimal at physiological flow rates according theory of filament surfing

Sperm of marine invertebrates have to find eggs cells in the ocean. Turbulent flows mix sperm and egg cells up to the millimeter scale; below this, active swimming and chemotaxis become important. Previous work addressed either turbulent mixing or chemotaxis in still water. Here, we present a general theory of sperm chemotaxis inside the smallest eddies of turbulent flow, where signaling molecules released by egg cells are spread into thin concentration filaments. Sperm cells ‘surf’ along these filaments towards the egg. External flows make filaments longer, but also thinner. These opposing effects set an optimal flow strength. The optimum predicted by our theory matches flow measurements in shallow coastal waters. Our theory quantitatively agrees with two previous fertilization experiments in Taylor-Couette chambers and provides a mechanistic understanding of these early experiments. ‘Surfing along concentration filaments’ could be a paradigm for navigation in complex environments in the presence of turbulent flow.     

Sperm-induced currents at fertilization in sea urchin eggs injected with EGTA and neomycin.

Membrane currents were measured in single voltage-clamped sea urchin eggs (Lytechinus pictus and Lytechinus variegatus) that were injected with either EGTA or neomycin and inseminated. Although egg activation and the fertilization calcium wave were prevented by injection of either of these compounds, sperm attached and still elicited inward currents. Sperm-induced currents in EGTA-injected eggs had an abrupt onset, quickly reached a maximum, and then slowly declined in amplitude. Sperm incorporation occurred readily in EGTA-injected eggs. Similar results were obtained with another calcium chelator, BAPTA. In neomycin-injected eggs, sperm-induced currents generally had an abrupt onset and, in contrast to EGTA-injected eggs, the currents usually cut off rapidly. Sperm failed to enter the neomycin-injected eggs and the duration of sperm-induced currents in neomycin-injected eggs was markedly dependent upon the voltage-clamp holding potential, with shorter duration currents occurring at -70 than at -20 mV. The lability of the initial interaction between sperm and egg at negative holding potentials may explain why activation often fails when the egg membrane is voltage clamped at these potentials (Lynn et al., Dev. Biol. 128, 305-323, 1988).     

Analysis of fertilization and polyspermy in serotonin-spawned eggs of the zebra mussel,Dreissena polymorpha

Eggs isolated from animals spawned with 10⁻³ M serotonin were inseminated with sperm concentrations ranging from 10³–10⁶ sperm/ml. Multiple sperm attached to the surface of the egg and sperm incorporation occurred within 3 min postinsemination (PI). Sperm mitochondria, centrioles, and flagellum were also incorporated. Incorporation was essentially complete by 6 min PI. In the egg cortex, the sperm head rotated 180°, and a rapid translocation of the sperm through the cytoplasm towards the egg interior began by 5–6 min PI. In heavily polyspermic inseminations, translocations of the sperm were either minimal or nonexistent. In monospermic eggs, nuclear decondensation occurred after translocation was complete, beginning by 9–10 min PI. A male pronucleus began to develop in the cytoplasm by 21 min PI and enlarged to 20 μm before fusing with the female pronucleus. Oscillation of the egg cytoplasm and mitotic spindle apparatus was observed immediately prior to cleavage. Cleavage occurred at 60 min PI. Sperm incorporation and pronuclear formation were confirmed with fluorescent and confocal microscopy using the DNA-specific dyes Hoescht 33342 and 7-aminoactinomycin D. In sperm concentrations >10⁴ sperm/ml, 26–76% of the eggs exhibited polyspermy. The high incidence of polyspermy suggests that rapid, effective blocks to polyspermy were not present or were ineffective in a significant proportion of serotonin-spawned eggs. © 1996 Wiley-Liss, Inc.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

FERTILIZATION IN OEDOGONIUM. I. PLASMOGAMY12

Events prior to, during, and immediately following plasmogamy have been investigated in Oedogonium cardiacum using combined techniques of light and electron microscopy. Maturation of the oogonium involves the formation of an oogonial pore and the differentiation of the single egg from the larger oogonial protoplast from which it is formed. The fine structure of the sperm cell at the time of plasmogamy is described as well as the nature of its entrance into the oogonium. Cinematographic films were used to analyze the movements of the spermatozoids prior to plasmogamy and, similarly, 26 complete acts of gametic fusion were recorded and analyzed. Prior to plasmogamy the flagella-bearing anterior extremity of the spermatozoid typically becomes elongate and is thereafter capable of flexible movements and rapid changes in shape which appear more or less independent of the rest of the cell. The sperm cell always makes initial contact with the egg surface by means of this agile, proboscis-like, anterior end. Contact results through a combination of thrusting movements of the entire sperm cell and rapid, lateral sweeping movements of its flagellated anterior extremity against the egg surface. Gametic fusion is initiated with violent, vibrational movements of the sperm cell accompanied by loss of its flagella. Apparent fusion of the gamete membranes unites their protoplasts by a narrow cytoplasmic bridge which gradually increases in size as the sperm cell cytoplasm flows into the egg. An average time of 30.5 sec was required for complete fusion as determined from 25 typical sequences of plasmogamy recorded cinematographically. Fusion occurs even more rapidly when diploid oogonia are substituted for daploid oogonia. The entire sperm cell, with the exception of the flagella, fuses with the egg during plasmogamy. The dissimilar gamete nuclei are clearly distinguished ultrastructurally in the binucleate fusion cell. Concentrations of sperm cell mitochondria and remains of the flagellar apparatus (but no flagella) are readily recognized in the fusion cell. The fate of these and other cytoplasmic constituents of the sperm cell is discussed. Immediately after plasmogamy, and prior to karyogamy, a thin, finely fibrous layer is formed us an investment exterior to the fusion cell. Karyogamy follows shortly after plasmogamy, and both events may take place within 15 min after mixing eggs and spermatozoids.     

Light and Electron Microscopic Studies on Fertilization of Pelmatohydra Robusta I. Sperm Entry to a Specialized Region of the Egg

Fertilization of a fresh water polyp, Pelmatohydra robusta , was studied by light and electron microscopy. A small depression was observed in the animal pole of the unfertilized egg. The egg pronucleus was always situated in close contact with the bottom of the depression. Microvilli which were covered with an egg coat consisting of filamentous components were observed on the egg surface. Microvilli and the egg coat were not detected on the surface of the depression. Sperm were associated with the egg plasma membrane and entered the egg only at the bottom of the depression. Excess sperm aggregated around the depression of inseminated eggs. After fertilization, the egg made a protrusion in the region where the egg pronucleus and sperm were in close contact with each other. A new egg coat was formed on the entire surface of the fertilized egg. The restriction of sperm-egg interactions to a specialized region of the hydra egg is discussed in connection with the micropyle of Pisces eggs and the animal dimple of Discoglossus (Anura) eggs.     

Hydrozoan eggs can only be fertilized at the site of polar body formation

Hydrozoan eggs are normally fertilized at the site of polar body formation. The female pronucleus is just under the cell membrane at this site. Sperm are attracted to the eggs and aggregate at this site. This paper demonstrates that this site is the only region on the egg surface where the sperm can fuse with the egg. This has been done by cutting unfertilized eggs into fragments containing the site of polar body formation and fragments without this region. Sperm were added to the fragments and their ability to be fertilized was assayed by noting whether or not they cleaved. Only fragments containing the site of polar body formation cleaved. The absence of cleavage in fragments lacking the site of polar body formation cannot be attributed to the inability of these fragments to attract sperm. Such fragments attract sperm for several hours while fragments which contain the site of polar body formation stop attracting sperm a few minutes after fertilization. Cytological studies of egg fragments which do not contain the site of polar body formation show that they do not contain sperm nuclei. The lack of cleavage in these fragments cannot be attributed to the lack of a female pronucleus. By using centrifugation it is possible to move the female pronucleus away from the site of polar body formation. By cutting these centrifuged eggs in an appropriate way it is possible to create egg fragments with the site of polar body formation that lack the female pronucleus and egg fragments that lack the site of polar body formation but contain a female pronucleus. Only fragments which contain the site of polar body formation can be fertilized.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

Something Darwin didn't know about barnacles: spermcast mating in a common stalked species

Most free-living barnacles are hermaphroditic, and eggs are presumed to be fertilized either by pseudo-copulation or self-fertilization. Although the common northeast Pacific intertidal gooseneck barnacle, Pollicipes polymerus, is believed only to cross-fertilize, some isolated individuals well outside penis range nonetheless bear fertilized eggs. They must therefore either self-fertilize or—contrary to all prior expectations about barnacle mating—obtain sperm from the water. To test these alternative hypotheses, we collected isolated individuals bearing egg masses, as well as isolated pairs where at least one parent carried egg masses. Using 16 single nucleotide polymorphism markers, we confirmed that a high percentage of eggs were fertilized with sperm captured from the water. Sperm capture occurred in 100 per cent of isolated individuals and, remarkably, even in 24 per cent of individuals that had an adjacent partner. Replicate subsamples of individual egg masses confirmed that eggs fertilized by captured sperm occurred throughout the egg mass. Sperm capture may therefore be a common supplement to pseudo-copulation in this species. These observations (i) overturn over a century of beliefs about what barnacles can (or cannot) do in terms of sperm transfer, (ii) raise doubts about prior claims of self-fertilization in barnacles, (iii) raise interesting questions about the capacity for sperm capture in other species (particularly those with short penises), and (iv) show, we believe for the first time, that spermcast mating can occur in an aquatic arthropod.     

Does egg competition occur in marine broadcast‐spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.     

Activity of sperm and fertility in the potato moth, Phthorimaea operculella

In this investigation, two kinds of sperm (apyrene and eupyrene) were found in the potato moth. At each mating, a single spermatophore containing both types of sperm was passed to the female. Sperm storage was observed in males in the duplex and in the females in the spermatheca. The fertility of eggs was greater than 90 per cent. Sperm survival in females was from one to 12 days after mating, as determined by egg hatching. Parthenogenesis was absent.