FERTILIZATION IN OEDOGONIUM. III. KARYOGAMY

Karyogamy is described in Oedogonium cardiacum from ultrastructural studies. Close proximity of the two gamete nuclei in the fusion cell is established by plasmogamy, whereas karyogamy appears to be initiated by multiple contacts formed between the outer membranes of the adjoining nuclear envelopes. Blebs of endoplasmic reticulum (ER) originate from the outer membrane of each nuclear envelope; these ER blebs presumably contact and fuse with the outer membrane of the nuclear envelope of the opposing nucleus. This is followed by the fusion of the inner membranes of the opposing nuclear envelopes, thereby resulting in a series of small connective bridges between the two gamete nuclei. It is estimated that in this manner 30–50 bridges are formed, perhaps many more. Several of these bridges enlarge relative to the others; one presumably becomes the major connection between the fusing nuclei. As it continues to enlarge, any organelles positioned between the fusing nuclei are pushed aside. There is also evidence, particularly in later stages of karyogamy, that the smaller connective bridges fuse to form larger ones. Temporary cytoplasmic channels often result at the juncture of fusion. In other instances, isolated inclusions of cytoplasm may be delimited by remnants of nuclear envelope deep within the developing zygote nucleus; these inclusions disappear with subsequent development. Throughout karyogamy the contribution of the male gamete nucleus is readily recognized by the characteristic appearance of its highly condensed chromatin. Ultimately, however, this distinction is lost and the content of the mature zygote nucleus assumes a more uniform appearance very similar to that of an egg nucleus. The complete process of fertilization in Oedogonium may occur within 15 min of mixing the spermatozoids with eggs.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.     

Renovation of the egg extracellular matrix at fertilization

Extracellular matrices are essential for cell survival and function. This is especially relevant for eggs, which establish a physical barrier at fertilization to protect a new embryo from additional sperm and pathogens. Formation of an extracellular matrix is most dramatic in sea urchins, in which fertilization was first observed in animals with the "sudden appearance of a perfectly transparent envelope" (A. Derbs, 1847). The process of assembling this extracellular "envelope" has been a topic of intense study ever since. Here we integrate the cellular and molecular events necessary to form this fertilization envelope within the first few minutes of a new embryo's life.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

FORMATION OF THE CORTICAL CONCAVITY AT FERTILIZATION IN THE SEA URCHIN EGG

During the initial stages of fertilization envelope elevation in eggs of Strongylocentrotus pur puratus and S. droebachiensis a large concavity of the egg cortex was observed in the light microscope. This concavity corresponded in shape and size with the elevating fertilization envelope. However, after the vitelline layers of eggs were disrupted and the eggs inseminated, the concavity failed to develop although the eggs were fertilized and developed normally. We propose that the concavity is formed owing to increased hydrostatic pressure within the perivitelline space. To further support this hypothesis we measured total egg protein secreted during fertilization, and found that 98% was retained within the perivitelline space. Furthermore, 80% of the total protein was contributed by the hyaline layer. Presumably, colloidal osmotic pressure and/or hydration of fertilization product, trapped beneath the fertilization envelope, is responsible for increased hydrostatic pressure within the perivitelline space, and therefore promotes not only fertilization envelope elevation, but the cortical concavity as well.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization

Unfertilized eggs of the rose bitterling (Rhodeus ocellatus ocellatus) were squeezed out of females that had an elongated ovipositor and were dechorionated mechanically with fine forceps in physiological saline. The dechorionated eggs were transferred into fresh water then inseminated at once by spermatozoa of the same species. A large number of spermatozoa was found on the surface of eggs that had not yet had cortical reaction following insemination. The surface of the naked eggs responded by formation of many small cytoplasmic protrusions (viz., fertilization cones) at sperm attachment sites. The formed fertilization cones were rosettelike structures formed by the aggregation of some bleblike swellings devoid of microvilli and microplicae. About 10 min after insemination, the fertilization cones retracted, but marks of their presence characterized by less microvilli and microplicae remained in the eggs 15 min after insemination. Many spermatozoa penetrated into the cytoplasm of each naked egg. The sperm nuclear envelope disappeared by means of vesiculation resulting from fusion of the inner and outer membranes. The sperm nucleus decondensed and developed into a larger male pronucleus. Smooth-surfaced vesicles surrounded the decondensing sperm nucleus and formed the new male pronuclear envelope. Sperm mitochondria and flagella were found in the egg 15 min after insemination. The response of the egg surface to sperm entry and pronucleus formation are discussed.     

Immunocytochemical localization of the 35-kDa sea urchin egg trypsin-like protease and its effects upon the egg surface

Trypsin-like protease in sea urchin eggs is thought to reside in cortical granules since it is secreted at fertilization and has been isolated with cortical granule fractions from unfertilized eggs. A 35-kDa serine protease has been purified from Strongylocentrotus purpuratus eggs by soybean trypsin inhibitor-affinity chromatography. For this report the protease was localized by immunocytochemistry before and after fertilization, and its potential biological activity was examined by application of the isolated enzyme to the unfertilized egg surface. The protease was localized on sections by immunofluorescence and immunoelectron microscopy, and was found to reside in the spiral lamellae of S. purpuratus cortical granules and in the electron-dense stellate core of Arbacia punctulata granules. At fertilization the enzyme is secreted into the perivitelline space and accumulates only very briefly between the hyaline layer and the nascent fertilization envelope. Shortly thereafter the enzyme is lost from the perivitelline space and immunological reactivity is no longer associated with the egg surface. The 35-kDa cortical granule protease has vitelline delaminase activity but does not appear to destroy vitelline envelope sperm receptors as judged by the fertility of protease-treated eggs.     

Artificial insemination using cryopreserved sperm in the silkworm,Bombyx mori

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Secretions from the female gametophyte and their role in spermatozoid induction in Cycas revoluta

In cycads, spermatozoids are released from pollen tubes and swim in fluid toward the archegonia. The source of this fluid was examined using Cycas revoluta Thunb. ovules placed in culture. Dissected female gametophytes just before fertilization produced copious fluid on their upper surface. The fluid first appeared around the archegonial chamber and then on the inside of the archegonial chamber. When this fluid was applied to dry turgid pollen tubes, they discharged spermatozoids 12 h later. The archegonial neck appeared as two semi-spherical swellings, whereas the four neck cells later became visible and they separated in a schizogenous manner. Many globose particles appear on the top of the archegonial neck cells when the fluid is present. The contents of pollen tubes, spermatozoids and surrounding liquid intermingle with the secreted fluid. The female gametophyte differs in ultrastructure during the stages before and after fluid secretion, the latter showing changes suggestive of fluid secretion from the female gametophyte.     

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.     

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca2+ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 microseconds resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca2+ through the voltage-induced pores. In a medium containing 100 microM Ca2+ and 45Ca2+ tracer, the voltage-treated eggs admitted 3.6 +/- 0.3 fmol Ca2+/egg within a few seconds. Untreated eggs took up only 1.0 +/- 0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca2+ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca2+ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca2+, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occurred only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca2+ to trigger cortical granule breakdown and formation of the fertilization envelope.     

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs.

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin

[³H]-dopamine ([³H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [³H]-DA uptake depended on time and [³H]-DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.     

Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin

Uptake and phosphorylation of externally supplied [3H]-thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 micrograms/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin-treated eggs increases greater than 50-fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]-thymidine triphosphate (3H-dTTP) resulting from an external supply of [3H]-thymidine is therefore equal in aphidicolin-treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]-thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H-dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 micrograms/ml. In the continuous presence of 5.0 micrograms/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.     

Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.     

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine- treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.