SPERM BUNDLE-FEMALE SOMATIC CELL INTERACTION IN THE FERTILIZATION PROCESS OF VOLVOX CARTERI F. WEISMANNIA(CHLOROPHYTA)1

We studied the fertilization reaction in Volvox carter f. weismannia (Powers) Iyengar. Tests for a sperm bundle chemoattractant produced by female spheroids were negative. The flagella of the female spheroid were identified as the site of sperm bundle binding. Treatment of female spheroids with trypsin or protease blocked sperm bundle binding, suggesting surface proteins are involved. Sperm bundle binding is not affected by female spheroid age up to 64 h after inversion of the female spheroid. Soybean trypsin inhibitor prevents fertilization pore formation. This suggests that a trypsin-like enzyme, released by the dissociating sperm bundle, is responsible for fertilization pore formation.     

Formation and structural organization of the egg�Csperm bundle of the scleractinian coral Montipora capitata

The majority of scleractinian corals are hermaphrodites that broadcast spawn their gametes separately or packaged as egg–sperm bundles during spawning events that are timed to the lunar cycle. The egg–sperm bundle is an efficient way of transporting gametes to the ocean surface where fertilization takes place, while minimizing sperm dilution and maximizing the opportunity for gamete encounters during a spawning event. To date, there are few studies that focus on the formation and structure of egg–sperm bundle. This study explores formation, ultrastructure, and longevity of the egg–sperm bundle in Montipora capitata, a major reef building coral in Hawai‘i. Our results show that the egg–sperm bundle is formed by a mucus layer secreted by the oocytes. The sperm package is located at the center of each bundle, possibly reflecting the development of male and female gametes in different mesenteries. Once the egg–sperm bundle has reached the ocean surface, it breaks open within 10–35 min, depending on the environmental conditions (i.e., wind, water turbulence). Although the bundle has an ephemeral life span, the formation of an egg–sperm bundle is a fundamental part of the reproductive process that could be strongly influenced by climate change and deterioration of water quality (due to anthropogenic effects) and thus requires further investigation.     

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm-incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate-containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.     

FLAGELLAR MOTILITY IS NOT INVOLVED IN THE INCORPORATION OF THE SPERM INTO THE EGG AT FERTILIZATION*

Cinemicrography of sea urchin fertilization reveals that the fertilizing sperm is one of the first sperm to attach to the egg. Just before the cortical reaction the fertilizing sperm ceases motility and then is incorporated into the egg without flagellar beating. The rate of incorporation is 5–11 μm/sec and is constant. Lytechinus pictus sperm rendered immotile by azide treatment can bind to and fertilize eggs but binding, and therefore fertilization, is blocked by azide treatment of Strongylocentrotus purpuratus gametes.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Egg jelly influences sperm motility in the externally fertilizing frog,Crinia georgiana

Recent in vitro fertilization studies have revealed female and male × female interaction effects on the probability of fertilization. These findings suggest a mechanism of cryptic female choice via sperm–egg interactions. The egg jelly of anuran amphibians contains proteins that facilitate the chemoattraction and binding of sperm for fertilization. Here we show that egg jelly also influences the onset of motility and swimming velocity of motile sperm in the frog Crinia georgiana. Moreover, we found significant among female variation in the effects of egg jelly on sperm motility. We discuss this finding with respect to male and female effects on nonrandom fertilization observed in this species.     

The reproductive system of Osedax (Annelida,Siboglinidae): ovary structure,sperm ultrastructure,and fertilization mode

Osedax is a genus of siboglinid annelids in which the females live on dead vertebrate bones on the seafloor. These females have a posterior end that lies within the bone and contains the ovarian tissue, as well as the “roots” involved with bone degradation and nutrition. The males are microscopic and live as “harems” in the lumen of the gelatinous tube that surrounds the female trunk, well away from the ovary. Females are known to spawn fertilized primary oocytes, suggesting internal fertilization. However, little is known about sperm transfer, sperm storage, or the location of fertilization, and the morphology of the female reproductive system has not been described and compared with the reproductive systems of other siboglinids. A 3D‐reconstruction of the ovisac of Osedax showed ovarian tissue with multiple lobes and mature oocytes stored in a “uterus” before being released through the single oviduct. The oviduct emerges as a gonopore on the trunk and travels along the trunk to finally open to the seawater as a thin cylindrical tube among the crown of palps. Light and transmission electron microscopy of mature Osedax sperm revealed elongate heads consisting of a nucleus with helical grooves occupied by mitochondria. In contrast to other Siboglinidae, Osedax sperm are not packaged into spermatophores or spermatozeugmata, and Osedax females lack a discrete region for sperm storage. Transmission electron microscopy and fluorescence microscopy allowed detection of sperm associated with ovarian tissue of the female ovisac of four different Osedax species. This provides the first evidence for the site of internal fertilization in Osedax. A heart body was found in the circulatory system, as seen in other siboglinids and some other annelids. The possible presence of nephridia in the anterior ovisac region was also documented. These morphological features provide new insights for comparing the regionalization of Osedax females in relation to other siboglinids.     

High fertilization success in a surface-brooding Caribbean gorgonian

Colonies of the Caribbean gorgonian Pseudopterogorgia elisabethae release eggs that are retained on the colony surface where they are fertilized and then develop. In December 2001, spawning on San Salvador Island, Bahamas, occurred over 6 d, with spawning by any one colony limited to 1-3 d. With the exception of the first and last days of the spawning period, fertilization success was high, often greater than 90%. Eggs collected in December 2001 had an overall fertilization success of more than 66%. At one site, the increase in fertilization after the first day of spawning correlated with male spawning, but male gonad index was a poor predictor of fertilization success. The number of male colonies close to a female was not correlated with fertilization success. Surface brooding is an efficient mechanism for "harvesting" sperm released upstream of female colonies. By maintaining their eggs at a single location, surface-brooding species can extend the period over which eggs are likely to encounter sperm. As a result, fertilization success is summed across the temporal variance in sperm availability, and the need for very high densities of sperm, with its concomitant risk of polyspermy, may be reduced.     

Differentiation of Reproductive Cells in Volvox carteri*

SYNOPSIS. The life cycle of Volvox carteri was studied in axenic culture using the NB-3 and the NB-7 strains isolated from Nebraska. Vegetative colonies of both strains contain 8–12 asexual reproductive cells (gonidia) which divide to form daughter colonies. During daughter colony formation, the reproductive cells of the daughters are delimited at an early stage of cleavage. Gonidia are delimited at the division from 16 to 32 cells, but eggs and male initial cells are not differentiated until the division of the 32-celled stage. In all instances the reproductive cells are the products of unequal cleavages. Male and female colonies are formed in separate clones. Female colonies contain approximately 20 eggs. Male colonies have approximately 50 male initial cells, each of which forms a sperm bundle containing 64 or 128 sperm. Sperm bundles penetrate female colonies and fertilize the eggs. Zygote formation, zygote germination, and the development of gone colonies is described. Sexual type was inherited in a 1:1 ratio. Male colonies appear spontaneously in the male strain, but female colonies were formed in the female strain only in the presence of a substance produced by colonies from male cultures. This female inducing substance is produced in male cultures primarily, if not exclusively, by male colonies rather than by vegetative colonies. The female inducing substance is heat labile and non-dialyzable. Activity is destroyed by Pronase, but not by trypsin, chymotrypsin or ribonuclease. Gonidia appear to be most susceptible to female induction during the early stages of their expansion prior to cleavage.     

Effects of motility inhibitors during sea urchin fertilization : Microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex, whereas microtubule inhibitors prevent pronuclear migrations

The sensitivity of specific stages of fertilization to microfilament inhibitors (cytochalasins B (CB), D (CD), and E (CE) and phalloidin) and to inhibitors of microtubule assembly (colcemid (CMD), colchicine (CLC), griseofulvin (GSF), maytansine (MAY), nocodazole (NCD), podophyllotoxin (PDP), and vinblastine (VB)) was investigated using differential interference contrast, time-lapse video microscopy of the sea urchin Lytechinus variegatus. Cytochalasins (CDCE>CB) will prevent sperm incorporation if added prior to or simultaneous with insemination. Sperm-egg fusion and the cortical reaction appear normal, but then the subsequent elevation of the fertilization coat lifts and eventually detaches the ‘fertilizing’ sperm from the egg plasma membrane. When the cytochalasins are added after fusion, the forming fertilization cone is rapidly resorbed, and the lateral displacement of the sperm along the egg cortex is terminated; the pronuclear migrations and mitoses occur normally though cytokinesis is never observed. Cytochalasin treatment before or within 2 min of insemination results in the development of aberrant egg cortices, whereas cytochalasin treatments after 2 min post-fusion have little effect. Phalloidin results in large and long-lasting fertilization cones and a retardation of the rate of sperm incorporation. Eggs exposed to any of the microtubule inhibitors 15 min prior to insemination will incorporate the spermatozoon, though the formation of the sperm aster and the accompanying pronuclear migrations are prevented. Interestingly, the final stage of sperm incorporation involving a lateral displacement of the sperm along the egg cortex is greater (27.1 vs 12.4 μm in controls) and faster (5.4 vs 3.5 μm/min in controls) in microtubule-inhibited eggs. GSF and VB, which readily permeate fertilized eggs, will prevent the formation of the sperm aster if added 3 min after sperm-egg fusion, they will prevent the migration of the female pronucleus if added 5 or 7 min after sperm-egg fusion, pronuclear centration if added 10 min post-fusion, and syngamy if added 12 min post-fusion. CLC- or CMD- treated eggs will develop normally if these drugs are photochemically inactivated with 366 nm light within 4 min post-fusion, arguing that sperm incorporation is completely independent of assembling microtubules. These results indicate that microfilament inhibitors will prevent sperm incorporation and the restructuring of the fertilized egg cortex, and that microtubule inhibitors will prevent the formation and functioning of the sperm aster during the pronuclear migrations; an interplay between cortical microfilaments and cytoplasmic microtubules appears required for the successful completion of fertilization.     

Suppression of photo-induced sporulation inTrichoderma viride by inhibitors

The mycelium ofTrichoderma viride grown in the dark under submerged conditions and transferred to membrane filters sporulated only after photoinduction. The optimum photoinduction of sporulation was reached when applying daylight for 3 min and near ultraviolet radiation (366 nm) for 10 to 30 sec. After the photoinduction pronounced synthesis of DNA, RNA and protein was observed. The photoinduced sporulation was partially or fully inhibited in the presence of phenethyl alcohol, actinomycin D, 5-fluorouracil, cycloheximide and ethidium bromide. The same inhibitors blocked also the photoinduced sporulation of surface growing colonies ofTrichoderma viride. Various inhibitors of synthesis of nucleic acids and protein, inhibitors impairing the function of membranes and certain other compounds were also effective. A part of the results presented here was included in the dissertation of J.S. and defended at the Slovak Polytechnical University in Bratislava.     

Effects of antibiotics on the life cycle ofNeurospora crassa

Some antibiotics and synthetic inhibitors affect, in several ways, the life cycle ofNeurospora crassa (germination of conidia → myceliar growth → formation of conidia). Bikaverin, cyanein, scopathrioin and phenethyl alcohol retard the germination of conidia, without inhibiting it completely. 5-Fluorouracil, ramihyphin A and zygosporin A (cytoohalasin D) do not inhibit the germination. Bikaverin brings about a thickening of the hyphae of growing mycelium. Ramihyphin A, cyanein, scopathricin and zygosporin A stimulate the ramification of hyphae while 5-fluorouracil and phenethyl alcohol do not affect the myceliar morphology apart from their inhibitory effect on growth. Actinomycin D, 5-fluorouracil, cycloheximide, ramihyphin A and partially also sodium iodoacetate inhibit to a different degree the photoinduced formation of conidia. The inhibition by 5-fluorouracil is very conspicuous when the agent is present during the photoinduction but considerably weaker when it is applied 2 h after the photoinduction.     

Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

Double fertilization in Gnetum gnemon (Gnetaceae): its bearing on the evolution of sexual reproduction within the Gnetales and the anthophyte clade

The fertilization process in Gnetum is critical to our understanding of the evolution of sexual reproduction within the Gnetales, a monophyletic group of nonfiowering seed plants that are the closest living relatives to flowering plants. Although much is known about the fertilization process in Ephedra, which is basal within the Gnetales, little is known about sexual reproduction in the derived sister groups Gnetum and Welwitschia. Ovules of Gnetum gnemon were collected at various stages after hand pollination and processed for light, fluorescence, and electron microscopy. Approximately 5 d after pollination, pollen tubes reach sexually mature female gametophytes, which are coenocytic. At that time, a binucleate sperm cell is found within each pollen tube. Within 7 d of pollination, double fertilization events occur when each of two sperm nuclei released from a pollen tube fuses with a separate, undifferentiated female nucleus within the free nuclear female gametophyte, which lacks differentiated egg cells. The products of double fertilization are two viable zygotes; endosperm is not formed. The lack of differentiated egg cells in Gnetum gnemon is unparalleled among land plants and the documentation of a regularly occurring process of double fertilization is congruent with the hypothesis that a rudimentary process of double fertilization evolved in a common ancestor of angiosperms and Gnetales.     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

The sperm-egg fusion in the nematode Parascaris equorum

Summary

The process of fertilization and the sperm storage in the female apparatus in Parascaris equorum is described in this paper. The sperm approaches the egg by means of pseudopodia containing bundles of microfilaments. The sperm and egg membranes fuse and the sperm penetrates progressively into the ovum. The egg and sperm plasma membranes and glycocalyces disappear at the point of fusion. At the end of fertilization, they are reformed at the egg's surface, while the egg and sperm chromatin begins to decondense. Spermatozoa are stored in the female apparatus prior to fertilization; here they come into contact with the epithelial cells of the spermatheca, protruding pseudopodia rich in microfilaments into the cellular body.     

Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes.

In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis in the presence of protein synthesis inhibitors (35 microM or 350 microM cycloheximide or a combination of inhibitors) (study 1); to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52% vs. 72%, P less than 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the oocyte.