Variation in penis morphology and mating ability in the acorn barnacle, Semibalanus balanoides

I examined variation in penis morphology of the acorn barnacle, Semibalanus balanoides, at different aggregation densities and at different levels of wave exposure. Barnacles in sparse, un-crowded aggregations had significantly longer penises than those from densely crowded groups, suggesting a response to increase the chance of reaching distant mating partners. Barnacles exposed to oceanic waves had penises with significantly greater basal diameter, possibly to strengthen the penis and retain function in turbulent conditions. I compared the percentage of individual barnacles with fertilized broods over a range of distances to their nearest possible mate in sites exposed to or protected from waves. As neighbor distance increased, the proportion of individuals with fertilized egg masses decreased in both wave-exposed and protected sites. However, at greater mate distances in the wave exposed sites, the proportion of individuals with fertilized eggs was significantly lower than the proportion in protected sites, indicating that exposure to waves hinders mating with neighbors at increasing distances. These results suggest that the intensity of mate competition may differ for barnacles between environments with different levels of wave exposure. These differences in male ability are predicted to alter relative sex allocation to male and female function.     

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%).     

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.     

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.     

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.     

Repeated Mating and Female Fecundity in the Simultaneously Hermaphroditic Land Snail Arianta arbustorum

Summary

To evaluate the influence of repeated mating on female fecundity in the simultaneously hermaphroditic, self-incompatible land snail Arianta arbus-torum, the number and size of clutches, egg size and hatching success of individuals from 3 populations were determined under conditions of isolation and grouping during one breeding season in a field cage experiment. Only adult snails which had mated and oviposited in the preceding year were used.

Sperm storage enabled isolated individuals to continue with the production of fertilized eggs. Snails kept singly or in groups differed neither in number of clutches laid nor in egg size. But isolated snails laid smaller clutches than did grouped snails. As a result isolated snails produced fewer eggs per breeding season. Hatching success varied greatly between populations and rearing conditions. In general, isolated snails had fewer hatchlings than grouped snails, indicating that individuals prevented from remating suffered a reduced fitness.     

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.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

Ascidian eggs block polyspermy by two independent mechanisms: One at the egg plasma membrane,the other involving the follicle cells

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm–egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 μM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 μM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm–egg fusion. Mol. Reprod. Dev. 48:137-143, 1997. © 1997 Wiley-Liss, Inc.     

Microsatellite markers and multiple paternity in a paternal care fish, Hexagrammos otakii

The fertilization success of sneaking behavior in a paternal care fish, Hexagrammos otakii, was estimated using microsatellites as genetic markers. Sneaking behavior was observed using a video camera placed near the nest of a breeding territorial male. Two egg masses fertilized by different patterns of sperm emission by the territorial male and sneaker males were examined for paternity discrimination. When a sneaker emitted a large amount of sperm after sperm emission by the territorial male, the sneaker fertilized more eggs than the territorial male. When the territorial male and a sneaker alternately emitted semen, the territorial male fertilized the most eggs. These results suggest that both the amount of emitted sperm and the timing of sperm emission are important factors in sperm competition. Received: November 18, 1999 / Accepted: January 17, 2000     

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.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

In vitro fertilization in ctenophores: sperm entry, mitosis, and the establishment of bilateral symmetry in Beroe ovata.

We have found ways to control in vitro fertilization in a ctenophore (Beroe ovata) for the first time. This is based on the existence of a partial block to self-fertilization at the time of gamete release which can be overcome by removal of the egg envelope. It has allowed us to exploit the excellent optical properties of Beroe eggs to make detailed observations on all events from sperm penetration or penetrations in these physiologically polyspermic eggs to first cleavage, and to extend our initial observations (Carré and Sardet, 1984). Sperm entry is characterized by local modifications of the egg cortex in a 70-microns zone around the penetration site or sites. Upon sperm entry, the egg surface contracts and relaxes locally, then a fertilization cone forms and disappears. These events are accompanied by localized exocytosis, growth of a ring of microvilli, thickening of the egg cortex, and gathering of mitochondria around the sperm pronuclei. The female pronucleus then migrates beneath the egg surface toward one or successive sperm pronuclei. The fusion of pronuclei, sperm and egg chromatin intermixing, and mitosis were also observed with exceptional clarity. Furthermore, we have noticed that the direction of the last trajectory of the female pronucleus tends to define the orientation of the mitotic spindle, and as a consequence the position of first unipolar cleavage furrow. This in turn determines the future sagittal plane of the embryo and of the adult B. ovata.     

Cytological evidence for self-fertilization in Lasaea subviridis (Galeommatacea: Bivalvia)

Summary

Isolated individuals of Lasaea subviridis simultaneously spawn both eggs and sperm. The sperm attach to the eggs by undergoing an acrosomal reaction and a male pronucleus is incorporated into the egg cytoplasm. Two reduction divisions associated with the production of two polar bodies occur before the first cleavage. These results are consistent with self-fertilization, but not with apomictic or meiotic parthenogenesis. The net fertilization efficiency of Lasaea subviridis natural populations is estimated at 99.93%.     

中华鲟初次全人工繁殖的特性研究

结合中华鲟研究所1994-1999年野生中华鲟人工繁殖亲本的生物学特性和2000-2008年野生成熟亲鱼人工繁殖的情况, 对子一代中华鲟初次全人工繁殖的特性进行了研究。结果表明: 虽然初次性成熟的子一代中华鲟个体均小于野生中华鲟的成熟亲本, 但性腺发育良好。经人工催产, 精子具有较高的质量(快速运动60s, 常温存活10min), 卵子也具有较高的受精率(71.4%)和孵化率(64.3%), 并成功的培育出了健康的子二代鱼苗1.8万尾。人工淡水条件下子一代中华鲟的全人工催产繁殖获得了成功, 证实了中华鲟可以在纯淡水中完成其生殖周期。研究结果对濒危物种中华鲟的物种延续具有重要现实意义。       

Fertilization and aqueous development of the puerto rican terrestrial-breeding frog,Eleutherodactylus coqui

The Puerto Rican tree frog, Eleutherodactylus coqui, has internal fertilization and direct development on land. In light of these reproductive adaptations, the events of fertilization and early development were studied. Cytological examination of just-fertilized eggs showed that sperm entry is restricted to about 10% of the surface of these large, yolky eggs, and all nuclear events of the first cell cycle occur near the animal pole. Although the oocytes have cortical granules, a number of polyspermic fertilizations were found. One clutch consisted of eggs with a high frequency of polyspermy and of normal development. This raises the possibility that normal development can occur despite multiple sperm entry, a situation not found in other anuran amphibians. With respect to saline requirements, the sperm and the embryo are similar to those in amphibians with external fertilization and aqueous development. Sperm motility was high in low-tonicity conditions, and the normally terrestrial embryo could develop completely from a fertilized egg to a froglet in a low-tonicity aqueous solution.     

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.     

UTILIZATION AND DEGREE OF DEPLETION OF EXOGENOUS SPERM IN THREE HERMAPHRODITIC SEA HARES OF THE GENUS APLYSIA (GASTROPODA: OPISTHOBRANCHIA)

Two series of experiments were conducted to examine the utilizationof exogenous sperm and their degree of depletion in three hermaphroditicsea hares, Aplysia kurodai Baba, 1937, A. Juliana Quoy &Gaunard, 1832, and A. parvula Guilding in MOrch, 1863. The firstseries of experiments was designed to determine whether onemating is enough for female partners to receive sufficient spermto fertilize at least one egg mass. For this purpose, isolatedindividuals were mated as females once with conspecific individualsand the viability of their subsequent eggs was examined. Whensuccessfully inseminated, individuals of all the three speciesreceived enough sperm to fertilize at least one egg mass, andthey continued to lay 3.6–8.5 viable egg masses. However,10–36% of the matings by these sea hares did not resultin any viable egg production by the female partner, indicatingthat these matings did not involve sperm transfer Such matingswithout sperm transfer tended to be shorter than matings withsuccessful sperm transfer The second experimental series wasconducted to examine whether sperm depletion actually occursor not in field-caught adults. For this purpose, adult individualscaught in the field were allowed to lay eggs under isolatedconditions without mating All the individuals of A. kurodaiand A. juliana stored enough sperm to fertilize at least oneegg mass. However, in A. parvula, 23–33% of individualslaid egg masses containing non-viable eggs at their first spawningafter isolation, indicating that these individuals were depletedin exogenous sperm (Received 17 July 1995; accepted 18 August 1995)     

Suitability of non-fertilized eggs of Homalodisca vitripennis for the egg parasitoid Gonatocerus morrilli

Gonatocerus morrilli (Howard) (Hymenoptera: Mymaridae) is an egg parasitoid used in California, USA to control glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Virgin GWSS females deposit non-fertilized eggs and mated females can exhaust sperm reserves for egg fertilization. However, nothing is known about Gonatocerus spp. performance when using non-fertilized GWSS eggs as hosts. Host age preference for oviposition and suitability of non-fertilized GWSS eggs as hosts for G. morrilli reproduction were investigated to determine whether non-fertilized eggs on sentinel plants could be used to monitor egg parasitoid populations. Gonatocerus morrilli parasitized all ages of GWSS eggs (1–8 days old) regardless if the host egg was fertilized or not. In choice tests (fertilized versus non-fertilized eggs), parasitoids failed to emerge as adults from non-fertilized eggs more often than from fertilized eggs. The results indicate that non-fertilized eggs were accepted by G. morrilli as suitable hosts for oviposition, but were less suitable for immature development compared to fertilized eggs.     

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.