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Experiments on cats established the presence of 3 primary cortical reaction zones responding to the stimulating action exerted on the receptor apparatus of the coronary vessels and pericardium. The first of them is located in the cruciate gyrus in the first zone of somatic sensation, partly overlapping the zone of the splanchnic nerve. The second zone is located in the ectosylvian gyrus in the second zone of somatic sensation. The site of the third one is the orbital region, in the zone of the vagus nerve.

Treatment of sturgeon eggs with the polyspermy-inducers urethane, ethyl ether, and acetone prolongs the latent period of the cortical reaction and apparently retards the actual secretion of the cortical granules. On activation of sturgeon eggs by pricking and of trout eggs by immersion in water, they lose their fertilizability at the moment of the discharge of cortical granules (the content of the alveoli) in the micropylar region. Before this no decrease in ability of the egg cytoplasm to accept a spermatozoon is observed. After removal of the perivitelline fluid (containing substances discharged from the cortical alveoli) penetration of numerous spermatozoa into activated trout eggs becomes possible. Perivitelline fluid of trout causes an agglutination of spermatozoa. The data obtained provide evidence that the block to polyspermy in fishes is a one-step process realized by means of the discharge of cortical granules (the content of the alveoli).

A study has been made of the fertilization of the lamprey egg, using Lampetra fluviatilis and L. planeri. No differences were found between these two species. 1. 1. The chorion of the egg has no micropyle, but spermatozoan entry is limited to the region of attachment of a tuft of fibrous jelly at the animal pole. 2. 2. At the end of a short swimming life, or on coming into contact with a solid surface, a very fine filament 50 μ long is extruded from the tip of the head. This reaction does not require the presence of eggs, egg water or of other adjuvant. 3. 3. This filament is the first part of the spermatozoon to come into contact with the egg and activate it. It also plays an essential part in getting the spermatozoon into the egg cytoplasm. 4. 4. There is no support for the view that the small cytoplasmic bleb which appears at the animal pole during the cortical reaction is a fertilization cone. The bleb appears after the entrance of a spermatozoon and no spermatozoon has been seen to pass through it into the egg. 5. 5. This study substantiates an earlier suggestion by Ballowitz on the role of the head filament and shows that fertilization in the lamprey egg closely resembles those non-chordates which have an acrosomal reaction.

Eggs of the sea urchin Arbacia punctulata were artificially activated with hypertonic seawater. The artificially activated eggs undergo the cortical reaction which is not distinguished by a wavelike progression as in the case of inseminated eggs. The cortical granules are released at random loci at the surface of the egg and result in spaces separated by large cytoplasmic projections. Unreacted cortical granules and ribosomes are found within the matrix comprising the large cytoplasmic projections. No "fertilization cone" is formed. The subsequent release of additional cortical granules results in the formation of a continuous perivitelline space, 15 min following activation. 85 min postactivation, an organization of annulate lamellae, endoplasmic reticulum of the smooth variety, and microtubules around a centriole is observed prior to nuclear division. Before the breakdown of the nuclear envelope a streak stage is formed. The streak is composed of a central core of annulate lamellae and is encompassed by endoplasmic reticulum and vesicular components. Condensation of chromatin is followed by the establishment of the mitotic apparatus. Centrioles were not found in the mature egg; however, they are present after activation prior to the first nuclear division, in the four-cell embryo, multicellular embryo, and at blastula. Artificially activated eggs have been observed to develop to the pluteus stage in more than 50% of the eggs treated.

Crystalline trypsin in 3 × 10−8 M concentration and higher, elicits fertilization membranes in the unfertilized eggs of Dendraster excentricus. These membranes are adequate in artificial parthenogenesis. If the action of trypsin on these eggs is continued for two or three hours the result is first, digestion of the membranes, followed later by reduction of the egg to amoeboid form. When fertilized, some of the partially digested eggs segment and form irregular cell masses, thus demonstrating that, in response to trypsin, there is first the cortical reaction giving rise to the fertilization membrane, and second, the progressive digestion and disintegration of the cytoplasm. Chymotrypsin causes rounding of the unfertilized eggs and, in rare instances, a few membranes, but the enzyme is not an adequate parthenogenetic agent. Fertilization of the egg renders the cytoplasm resistant to trypsin. The facts lead to the suggestion that fertilization liberates trypsin inhibitors in the cytoplasm.

Experiments in which oocytes were inseminated with heterologous spermatozoa showed that Qocytes are susceptible to the same species of sperm, as are the ripe eggs. Immediately after the spermatozoon has entered an egg or an oocyte, material appearing dark in the phase contrast microscope is ejected from the maternal nucleus; a ring-shaped corona is formed around the nucleus. No aster formation takes place in oocytes which have not yet developed the cortical reaction.

Eggs of the sea urchin, Arbacia punctulata, treated with 3% urethane for 30 sec followed by 0.3% urethane and inseminated are polyspermic and fail to undergo a typical cortical reaction. Upon insemination the vitelline layer of urethane-treated eggs either does not separate or is raised only a short distance from the oolemma. 1–6 min after insemination, almost all of the cortical granules remain intact and are dislodged from the plasmalemma. Later (6 min to the two-cell stage) some cortical granules are released randomly along the surface of the zygote. Not all zygotes show the same degree of cortical granule dehiscence; most of them experience little if any granule release whereas others demonstrate considerably more. The thickness of the hyaline layer appears to be directly related to the number of cortical granules released. Subsequent to pronuclear migration, several male pronuclei become associated with the female pronucleus. Later the male and female pronuclear envelopes contact and the outer and the inner laminae fuse, thereby forming the zygote nucleus. The male pronuclei remaining in the cytoplasm increase in size and progressively migrate to, and fuse with, the zygote nucleus. By 60 min some zygotes appear to contain only one large zygote nucleus which subsequently enters mitosis. Other zygotes possess a number of male pronuclei which remain unfused, and later these pronuclei along with the zygote nucleus undergo mitosis. There does not appear to be a direct relation between the number of cortical granules a zygote possesses and the above mentioned dichotomy.

THE prevention of polyspermy in the sea urchin egg is a two-stage process: a rapid reduction in the chances of refertilisation—the ‘fast block’1,2, which seems to be electrical in nature3—and a slower block that coincides with elevation of a morphological barrier, the fertilisation membrane. The fast block has many features in common with the action potential of excitable tissues, while elevation of the fertilisation membrane seems to involve a classical exocytotic reaction similar to that found at nerve terminals. Lying immediately beneath the plasma membrane of unfertilised eggs is an array of vesicles called cortical granules each about 1 µm in diameter. Shortly after fertilisation the contents of these granules are expelled, the granule membrane becoming incorporated into the plasma membrane of the egg. The trigger for initiating the cortical reaction seems to be a rise in intracellular ionised calcium. Thus granule release can also be promoted by the ionophore A23187 (ref. 4) and measurements in aequorin-loaded fish and sea urchin eggs5,6 show that exocytosis brought about by both sperm and ionophore is associated with a rise in intracellular ionised calcium. The mechanism of exocytosis in any system is unknown and the cortical granule reaction in sea urchin eggs seems to provide an excellent experimental preparation for its investigation. Here we examine the influence of metabolic poisons and calcium on the cortical reaction. In the presence of ATP exocytosis is activated by micromolar concentrations of calcium; but in the nominal absence of ATP the exocytotic process slowly becomes refractory to calcium.

SEA urchin eggs remain monospermic during fertilisation by excluding the entry of all but one sperm. Two mechanisms have been proposed to account for this exclusion. The first is a rapid alteration of the egg surface, lowering its receptivity to sperm, which occurs within a few seconds of successful contact of the fertilising sperm; this is the so-called fast block1. This block prevents or reduces further sperm entry for the next 30 s, until the time of the cortical reaction, which establishes the second and permanent block to polyspermy. This permanent or cortical block results from the release of cortical granule proteases, elevation of the fertilisation membrane, and subsequent detachment of sperm. The existence of a cortical block has been well established by a number of investigators2–4. Although the concept is intuitively compelling, the existence of a fast block has been questioned5,6, and it is supported by only one series of experiments1.

L'auteur a fait une etude microspectrophotometrique des cellules dePurkinje du rat blanc adulte. La courbe de distribution des frequences montre une remarquable dispersion des valeurs d'extinction cytoplasmiques a 2650 A et 2800 A, avec un groupement des frequences plus hautes vers les valeurs basses. L'etude des spectres d'absorption a demontre un maximum d'absorption des nucleotides et un maximum proteique, meme dans le cytoplasme des cellules moins absorbantes.

Fusion of the cortical granules with the oolemma and a discharge of their contents into the perivitelline space occurs when the sperm reaches the vitellus (Szollosi, 1967; Piko, 1969). Studies with hamster gametes in vitro have shown that the trigger for this so-called cortical reaction is fusion between the gamete membranes and that simple contact is not sufficient (Gwatkin et al., 1976b). Penetration of the sperm into the vitellus is not needed, since it was found that capacitated spermatozoa would induce the reaction even after they were frozen and thawed so as to prevent penetration. The cortical reaction is propagated around the egg from the point of association of the fertilizing sperm with the vitelline membrane (Braden et al., 1954).

Publisher Summary This chapter discusses the biochemical mechanisms of gamete interaction. The chapter focuses on three principal subjects: (1) the effect of the egg on the sperm; (2) the contact between sperm and egg; and, (3) the effect of the sperm on the egg. The most dramatic morphological response of the sperm to the egg is the acrosome reaction, in which a change that is a prerequisite for fertilization occurs in the head of sperm from many species. In mammals, sperms undergo a process known as capacitation before they respond to the egg. The moment of union of sperm and egg is the most dramatic of the events surrounding fertilization. The locus on the egg for interaction with sperm varies from species to species. Several enzymes have been implicated in the process of sperm–egg interaction. These enzymes play a role in sperm–egg membrane fusion, for it could lead to a transient state of membrane instability. The most dramatic effect of fertilization on the egg surface is the induction of the cortical reaction. The properties of the egg plasma membrane change enormously after cortical granule exocytosis.

We have examined the formation of the fertilization envelope in the lobsters Homarus americanus and H gammarus. Oocytes were fixed for electron microscopy either in the ovary or following extrusion from the gonopore. Mature ovarian oocytes are surrounded by a coat (envelope 1), which is comprised of small electron-dense granules and structures resembling "bottlebrushes." At least part of this coat is synthesized by the follicle cells of the ovary. The cortex of ovarian oocytes contains four types of vesicles that we refer to as high-density vesicles (HDV), low-density vesicles (LDV), moderately dense vesicles (MDV), and ring vesicles (RV). Oocytes that were electrically extruded from the gonopore and fixed immediately had an envelope identical to that of ovarian oocytes. The cortex of gonopore oocytes contained the four types of vesicles found in ovarian oocytes. When unfertilized gonopore oocytes were allowed to incubate in sea water, the oocyte cortex appeared unaltered, but envelope 1 swelled and the bottlebrushes dispersed. When recently fertilized oocytes were fixed during natural spawning or following in-vitro fertilization, each type of vesicle was released in sequence from the cortex of the oocyte. The contents of the HDV and LDV appeared first in the perivitelline space, but their fate could not be determined at later times. The ring-shaped elements of the RV and the moderately electron-dense material of the MDV were released exocytotically somewhat later; these materials coalesced in the perivitelline space to form a new coat (envelope 2). Envelope 1 subsequently condensed to its original thickness and appeared firmly attached to envelope 2. Our results show that the fertilized lobster egg is surrounded by two discrete coats. The outer coat, which is formed in the ovary, undergoes a swelling/condensation cycle at spawning. The inner coat originates from a complex cortical reaction. Together these coats comprise the fertilization envelope of the lobster egg.

Eggs, embryos and larvae of the intertidal sea anemone Actinia fragacea were obtained from spontaneous spawnings in the laboratory and have been examined by scanning and transmission electron microscopy. The eggs average 150 μm in diameter and are covered by tufts of large microvilli known as cytospines, but are not surrounded by a jelly layer or a vitelline coat. The cortical layer of the egg contains large numbers of dense, homogeneous cortical granules. The surface layers of cleavage and blastula stage embryos are similar in composition to those of unfertilized eggs in that the cytospine tufts remain intact and the number of cortical granules remains apparently undiminished. No major discharge of cortical granules indicative of a cortical reaction can have occurred. During gastrulation, many embryos take up large numbers of sperm by a process resembling phagocytosis. These sperm undergo breakdown in the superficial regions of the embryos. The cortical granules persist well into larval life, and their function is unknown.

International audience; We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.

Both isotopic and microelectrode studies reveal a significant Ca2+ influx at fertilization which if freely distributed in the cytoplasm would equal 1–2 × 10−5 M. The role, if any, of this influx is disputed. We have attempted to reevaluate contradictory findings by others on this role. Our results with Strongylocentrotus purpuratus and Lytechinus pictus eggs, assessing fertilization with acrosome-reacted sperm in EGTA-buffered media (free [Ca2+], 4.4 × 10−8 M) indicate that exogenous Ca2+ is not required for fertilization and subsequent cleavage. The contradictory findings by others may have resulted from reduced fertilizability in Ca2+-free seawater, which can be circumvented by higher sperm concentration and by a sensitivity to temperature in Ca2+-free medium, which can be bypassed by carrying out fertilization at lower temperature. Also consistent with the absence of a requirement for this Ca2+ influx, we found that Ca2+ uptake can be induced in eggs by depolarizing the membrane with high [K+], but there is no resultant activation of egg metabolism. Under our conditions for fertilization in Ca2+-free media, there is no effect on the block to polyspermy but the initiation of the cortical reaction may be delayed. The data support the hypothesis that sperm induce release of Ca2+ from intracellular stores, perhaps by affecting an equilibrium between Ca2+ sequestration and Ca2+ release.

The previous chapter dealt with mechanisms by which the invertebrate sperm responds to the egg, first undergoing the acrosome reaction and then binding to it before gamete membrane fusion. The fusion of sperm and egg is catastrophic for both the organism and the species. A new individual is formed with a reassortment of meiotic products that constitutes a new roll of the evolutionary dice. The mechanism of formation of the zygote has received increasing attention over the past decade with the application of modern techniques in molecular and cellular biology. In this chapter I will focus on some recent insights concerning several phenomena associated with activation of the sea urchin egg after fertilization. Work in this field occurs in the context of an excellent scientific literature that dates back well over a century; I shall try to point out some of the classical antecedents of the modern study of fertilization mechanisms.

For the first time, we have shown with appropriately labelled lectins that fucosyl- and sialyl-rich glycoconjugates are released into the perivitelline space of the mouse oocyte after activation by the fertilizing spermatozoon or artificial activation by the calcium ionophore A23187 or ethanol. The glycoconjugates show a punctate distribution over the oocyte surface except for the microvilli-free area overlying the second meiotic spindle from which they are absent. Their appearance in the perivitelline space is associated with the release of the cortical granules suggesting that they represent part of the cortical granule exudate. Soon after the glycoconjugates appear, they begin to aggregate. The process continues until the beginning of cytokinesis at first cleavage when a single large aggregate is found within the cleavage furrow. Most of the labelled glycoconjugates disappear by the late 2-cell stage and no evidence was found for their presence during the later preimplantation period. This technique is suitable for monitoring the kinetics of the cortical reaction in mammalian oocytes and investigating the importance of the glycoconjugates in early preimplantation period.

The argon laser was found to be suitable for pre-pain and pain stimulation. The visible (488 and 515 nm) argon laser light penetrates the skin and is absorbed by melanin and haemoglobin. Argon laser stimuli of different intensities were perceived differently, and could be classified into 3 pre-pain and 3 pain classes. The pre-pain sensations were either perceived as warmth, weak pin prick, or weak pin prick followed by warmth. The pain sensations were perceived as painful pin pricks of different intensities. Single cortical responses to argon laser stimuli were averaged selectively, according to this perceptual classification, and characteristic pre-pain and pain related cortical responses were recorded. The sensation of warmth was related to a late cortical deflection, 700–800 ms after stimulus onset, whereas the pin prick related response appeared with a latency of 300–400 ms. Pre-pain related responses were only recorded when selective averaging according to perception was used. The amplitude of the selectively averaged pain related cortical responses correlated with the subjective sensation of pain. Selective averaging of pre-pain and pain related single responses may prove useful in studying the cortical projection of different perceptions or modality patterns, and to investigate the function of the thermal and nociceptive pathways and their interactions.

Because of the limited number of eggs available for experimentation, little is known of the metabolic changes which follow fertilization in mammals. However, considerable knowledge has been obtained with echinoderm eggs (Monroy, 1965, 1973; Epel, 1975).

Experiments involving injections of cytoplasm from the cleaving embryos of Rana temporaria and Acipenser stellatus into fully grown oocytes of the same species and of Xenopus laevis, show that at all mitotic phases, and throughout the period of synchronous cleavage divisions, the cytoplasm is characterized by high activity of the germinal vesicle breakdown factor. This activity decreases during nuclear division desynchronization and drops sharply after the mitotic index fall upon blastulation. Aside from germinal vesicle breakdown in the oocytes, the A. stellatus embryos' cytoplasm also induces development of a cortical reaction capacity.

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca 2+ -free sea water (a treatment that bypasses the cortical reaction and the Ca 2+ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.

The events which surround fertilization and ultimately result in the beginning of the developmental program are referred to as “egg activation”, as attested by the title of this chapter. However, the definition of activation is “to make active” and the first item I discuss is whether this term is appropriate for what occurs at fertilization. I do not ask this to be pedantic or pugnacious, but because a search for a more appropriate term might lead to a better definition/description and understanding of fertilization.

Fine structural changes in the egg and sperm are described during gamete interaction in Oikopleura dioica, an appendicularian tunicate. The unfertilized egg has a vitelline layer 80 nm thick and a perivitelline space about 5 μm wide. In the peripheral cytoplasm are a few cortical granules 0.6×0.7 μm in diameter and areas rich in parallel cisternae of rough endoplasmic reticulum alternating with areas rich in long mitochondria. In the deeper cytoplasm the predominant organelles are multivesicular bodies. From 25 s to 60 s after insemination, the egg transiently elongates, although with no obvious cytoplasmic rearrangement, and the egg surface becomes bumpy. During this interval sperm enter the egg, and the cortical granules undergo exocytosis. After expulsion into the perivitelline space, the cortical granule contents do not appear to change their shape or blend with the vitelline layer, which neither elevates further nor loses its ability to bind sperm. On encountering the egg, the sperm undergoes an acrosome reaction involving exocytosis of the acrosome and production of an acrosomal tubule. The acrosomal contents bind the sperm to the vitelline layer, and the posterior portion of the acrosomal membrane and the anterior portion of the nuclear envelope evaginate together to form an acrosomal tubule, which fuses with the egg plasma membrane to form a fertilization cone. By 45 s after insemination, the sperm nucleus, centriole, mitochondrion and at least the anterior portion of the axoneme are within the fertilization cone. By 60 s sperm entry is complete. In having eggs with a cortical reaction and sperm with an acrosome reaction, O. dioica resembles echinoderms and enteropneusts and differs markedly from ascidian tunicates, which lack both these features. The relatively unmodified pattern of gamete interaction in O. dioica in comparison with the highly modified pattern in ascidians is difficult to reconcile with the neoteny theory that appendicularians have evolved via ascidian ancestors. The present results are more consistent with the idea that an appendicularian-like ancestor gave rise to ascidians.

Fertilization of urodele amphibians is physiologically polyspermic. These amphibians lack sperm entry blocking mechanisms at the egg surface, such as a cortical reaction or a membrane depolarization. Although, egg jelly is necessary for sperm capacitation, a late block to sperm entry does occur about 30 min after fertilization at a precise interface between jelly layers. The jelly is secreted by oviductal cells. In order to investigate its role in fertilization, we studied some biochemical properties of the oviductal secretions of eight species. 1) In double diffusion experiments on agarose plates, some components secreted by the anterior and the middle parts of the oviduct interacted together and formed precipitin lines. This reaction might be responsible for the formation of the dense zone that delimits the capsular chamber. 2) A hemagglutinating activity was found in the anterior or in the posterior part of the oviduct depending on the species. A 18K or 26K lectin was purified respectively from the oviduct of Ambystoma mexicanum and Pleurodeles waltl. In both species, the site where the late block to sperm entry is operative was spatially related to the location of the lectin in the jelly. However, sperm in contact with the purified lectins did not undergo any visible morphological change.

The zona pellucida (ZP) is a specialized extracellular matrix surrounding the mature oocyte. Specific functions ascribed to the ZP include sperm-binding, induction of the acrosome reaction, and involvement in the "slow block" to polyspermy. We have examined the ZP from a variety of human eggs recovered from follicular aspirates. Mechanically isolated ZP were heat-solubilized, iodinated, and characterized by SDS-PAGE. Under nonreducing conditions, the human ZP is characterized by two species of 92-120K (ZP1,2) and 57-73K (ZP3). After reduction, ZP1,2 separates into two components of 97K (ZP1) and 82K (ZP2), with ZP3 at 70K. Under reducing conditions, ZP specifically isolated from fertilized eggs reveals approximately equal proportions of all three iodinated ZP molecules. However, ZP specifically isolated from fertilized eggs reveals a substantial disappearance of ZP1. The ZP from unfertilized eggs does not display this modification of ZP1, even when solubilized zonae are co-incubated with sperm or sperm lysates. We suggest that this molecular change in ZP1 is a direct result of the egg cortical reaction. This demonstrates, for the first time, structural changes in the human ZP directly associated with fertilization. In additional studies, we have demonstrated that capacitated human sperm can be saturated with heat-solubilized ZP, demonstrating the presence of human ZP-binding proteins, and that the binding characteristics of zonae isolated from nonfertilized and fertilized eggs differ. Human sperm proteins having an affinity for solubilized zonae have also been identified at 16K, 18K, 19K, 35K, and 60K.

Cytochalasin B (CB) (2 × 10−6 M) prevents the incorporation of sperm into the eggs of Lytechinus pictus and Strongylocentrotus purpuratus as judged by light and transmission electron microscopy (TEM). At lower concentrations of CB (2 × 10−7 M), sperm are successfully incorporated into the egg, but their migration in the area of the egg cortex is impaired. The site of action of CB on the sperm may be on the initial rotation of the sperm nucleus in the cortex; the subsequent migration is not affected by CB. Although sperm incorporation is prevented at the higher CB concentrations, the eggs become activated—as judged by cortical reaction, increased protein synthesis and increased respiration. These findings raise the concept that egg activation by sperm could result from some pre-fusion event and hence that sperm-egg fusion would not be a prerequisite for the triggering of development. An alternative hypothesis is that fusion occurs between the acrosome process membrane and egg membrane, but since CB has destroyed the integrity of the cortex actin, the fusion bridge is so weak that it cannot be maintained without some contractile or cytoskeletal support by the cortex. The sperm may activate the CB-treated egg in the same manner as pricking with a microelectrode sometimes does.

Cortical granules in the mature, unfertilized egg of the zebra danio (Brachydanio rerio) are spherical, membrane-bound organelles measuring 3 to 22 μm in diameter and arranged in irregular rows beneath the plasma membrane. Two types of cortical granules are identified in thin sections viewed under the transmission electron microscope: “dark” type (electron-dense core) and “light” type (electron-lucent core). Cytochemically, glycoproteins and acid mucopolysaccharides are constituents of the cortical granules. Differences in staining reactions with Alcian blue and toluidine blue solutions also reveal the presence of two types of cortical granules in the danio egg. The “dark” type of cortical granule stains strongly with both dyes and shows a single, central core surrounded by a zone of lighter, particulate material. The “light” type of cortical granule stains faintly with both dyes. Differences in staining responses are not related to the size of the cortical granules. Analysis of the cortical granule reaction in inseminated eggs by light microscopy reveals that breakdown and discharge of cortical granules begin after 30 seconds postactivation. Since the chorion is lifted away from the egg surface by this time, the initial elevation of this membrane occurs independently of the onset of cortical granule exocytosis. The breakdown and discharge of cortical granules is a prominent feature of egg surface activity between 1 and 3 minutes postactivation. The cortical granule reaction is completed by 5 to 6 minutes postactivation. Scanning electron microscopy has provided an unusual opportunity to observe in detail cortical granule exocytosis and the reorganization of the egg surface during the cortical reaction. Cortical granule exocytosis consists of the following sequence of morphological stages: elevation of the cortical granule above the egg surface, rupture of the membrane covering the cortical granule, and discharge of central and peripheral constituents of the cortical granule into the perivitelline space. Rupture of the cortical granule follows contact and fusion between perigranular and egg plasma membranes and is effected through the coalescence of fissures that appear in the egg surface between the equator and the top of the elevated granule. Our SEM and TEM findings suggest strongly that the mosaic surface formed upon cortical granule exocytosis is temporary and that perigranular membranes have limited residence in the egg surface. Morphological evidence presented here is consistent with the view that the zebra danio egg accommodates the excessive membrane introduced into its surface during the cortical reaction by endocytosis. It is proposed that perigranular membrane is retrieved continuously after exocytosis but is visible morphologically as a two-step process. First, perigranular membrane is removed from the egg surface into the cortical cytoplasm in the form of vesicles (0.5–1.5 μm in diameter) pinched-off from the base of the cortical crypt. Second, localized folds of the egg plasmalemma overgrow and replace, perhaps through contractile activity, remaining perigranular membrane at the egg surface.

Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5–4.5 μ M free calcium 45–60 sec after activation and to last 2 3 min, assuming equal Ca 2+ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zerocalcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for observation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 18 μ M Ca 2+ . Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.

Relatively little is known about egg activation in the Crustacea, with the exception of morphological observations on the resumption of meiosis and the cortical reaction. Morphological analyses have demonstrated that the resumption of meiotic maturation is initiated by seawater contact in some decapod species. In the prawn Palaemon serratus, meiotic resumption of oocytes which are spawned at first meiotic metaphase, depends on the presence of external Mg2+ but not on external Ca2+ (Goudeau and Goudeau 1986). In Penaeid shrimp, meiotic resumption of oocytes, which are spawned at late prophase or early metaphase, requires Mg2+ when the gametes are fertilized and both Mg2+ and Ca2+ if unfertilized (Pillai and Clark 1987). In crabs, spawned oocytes are in first meiotic metaphase and also resume meiosis upon exposure to seawater. The process requires only 30–50 µM Ca2+ in Mg2+ free artificial seawater (ASW) (unpublished results). With respect to the cortical reaction, cytological observations have permitted the detection of a sperm-dependent cortical vesicle exocytosis in barnacle eggs (Klepal et al. 1979). In Penaeid oocytes, a specific release of jelly components, initiated by spawning and originating from extracellular crypts formed by invaginations of the plasma membrane, has been observed. Jelly components undergo an enzyme-mediated transition from a heterogeneous to homogeneous state, which is dependent on both Mg2+ in seawater and a protease (Clark et al. 1974; Clark and Lynn 1977; Clark et al. 1980; Clark et al. 1985; Lynn and Clark 1975). Also in Penaeid shrimp, the formation of a “hatching” envelope is induced by contact with seawater, requiring only external Mg2+ when the eggs are fertilized, and both Ca2+ and Mg2+ when they are not (Pillai and Clark 1987). Finally, a complex cortical reaction has been described in the eggs of crabs (Goudeau and Lachaise 1980 a,b; Goudeau and Becker 1982), and lobsters (Talbot and Goudeau 1988). The cortical reaction in crab eggs is a two-step phenomenon that consists of I) the exocytosis of a fine granular material, elicited by contact with seawater (Goudeau and Goudeau 1985), and 2) a slow and long-lasting exocytosis of ring-shaped elements, which is sperm-dependent and leads to the elaboration of a thick extracellular capsule (Goudeau and Lachaise 1980b; Goudeau and Becker 1982). In prawn eggs, the cortical reaction requires external Mg2+, and is independent of fertilization (unpublished results).

A scanning and transmission electron microscope study has been made of the cortical alveoli of the egg of Fundulus heteroclitus. The study includes both unactivated eggs and fertilized eggs fixed at intervals of 1 second to 10 minutes after insemination. The alveoli appear to vary considerably in size, in contents, and in morphological aspects of their breakdown. As it undergoes dehiscence, each vesicle may form one or several openings in the egg surface; dense granules and particulate, fibrous, or membranous material, apparently in any combination, are liberated to the nascent perivitelline space. It appears that much of the excess membrane externalized during the reaction is strung out in threads and probably lost to the perivitelline space. The evidence does not suggest that the excess membrane either “dissolves” or is retrieved by the egg cytoplasm. That part of the cortical vesicle membrane which remains continuous with the oolemma gradually becomes microvillous and loses its morphological identity. Granules and particulate matter, presumably liberated from the cortical alveoli, are seen adhering to the inner surface of chorions removed from activated eggs. The micropyle appears to be sealed with similar material. Supernumerary sperm are observed inside the chorion in some instances. The cortical reaction appears to play a secondary role in the prevention of polyspermy and to be somehow related to the subsequent formation of a normal embryonic blastodisc.

Studies on the activation of development at fertilization of the sea urchin egg have suggested that increases in free calcium and increases in intracellular pH are essential steps in triggering cell division (Whitaker and Steinhardt 1982; Steinhardt 1982). To link an ionic change to a subsequent event, such as cell division, it is necessary to demonstrate three things. First, one must be able to show that the ionic change occurs naturally and always accompanies the event in question. Second, artificially inducing the ionic change should lead to the induction of the event. Third, blocking the ionic change to the natural stimulus should block the subsequent event. All three conditions have been demonstrated for linking increases in free intracellular calcium and increases in intra-cellular pH to the activation of cell division at fertilization in the sea urchin egg.

International audience; Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.

EXPOSURE of unfertilised sea urchin eggs to NH4Cl and other weak bases such as procaine or nicotine results in the activation of some of the events which normally follow fertilisation, such as the acceleration of protein synthesis and the initiation of DNA synthesis1,2. The initial events of normal fertilisation, for example, the cortical reaction and respiratory burst, are bypassed. It has been shown that fertilisation triggers a Na–H ion exchange that results in an increase in the intracellular pH and a decrease in the pH of the surrounding seawater3. There is a similar acidification of the seawater and increase in intracellular pH when sea urchin eggs are exposed to NH4Cl or procaine. This led to the suggestion that the pH changes induced by weak bases were mediated by a mechanism similar to the Na–H ion exchange. However, it had been proposed that NH4Cl worked by a simple passive diffusion mechanism, where the uncharged species of weak base diffuses into the egg and picks up an H+ ion, resulting in an increase in the intracellular pH (ref. 4). The important distinction between these two mechanisms is that if weak bases are working by a passive diffusion mechanism, bypassing the Na–H ion exchange, then their action gives direct support to the hypothesis that an increase in the intracellular pH plays a part in the onset of biosynthetic activities after fertilisation. We show here that weak bases bypass the Na–H ion exchange, cause both the internal and external pH changes directly by their passive diffusion into the egg, and that the initial increase in the rate of protein synthesis is proportional to the amount of weak base entering the egg.

Fertilization of the egg is an all-important event for the life of all organisms that reproduce sexually. Not only does it start the machinery for embryonic development, but it also has fundamental genetic consequences: the diploid (2n) state is restored and the paternal hereditary characters are introduced into the fertilized egg (also called the zygote). In most species, the sex of the future adult is established at fertilization.

In the starfish Astropecten aurantiacus the acrosome reaction occurs when the spermatozoon contacts the outer surface of the jelly layer. A long thin acrosomal filament is extruded from the anterior region of the spermatozoon and establishes contact with the oocyte surface. This latter interaction initiates the movement of the spermatozoon to the oocyte surface, formation of the fertilization cone and the cortical reaction. The first detectable electrical change across the oocyte plasma membrane during interaction with the spermatozoon is the fertilization potential (FP) which occurs simultaneously with the cortical reaction. The FP is probably the electrical result of the modification of the oocyte plasma membrane during cortical exocytosis. There are no primary step-like depolarizations during fertilization of starfish oocytes, which contrasts with the situation in sea urchin eggs [see 13]. We suggest that the difference in electrical response to fertilization of starfish oocytes and sea urchin eggs may be attributed to the location of the acrosome reaction in these animals and not to their different meiotic states.

The ova of Sicyonia ingentis lack cortical vesicles at the time of spawning. Within 30 min post-spawning, two populations of cortical vesicles are organized in the ooplasm which, during cortical vesicle exocytosis (cortical reaction), successively release two morphologically different exudates. The first type of cortical vesicles (dense vesicles) appears to be derived from the Golgi complexes after spawning. The second type (the ring vesicles) is formed by the fusion of asternal elements which contain loosely packed ring-shaped structures that are present in the unactivated ova. During exocytosis of the dense vesicles an electron dense material is released which coalesces with the surface coat of the ovum to form a thin hatching envelope which eventually lifts from the ovum's surface. Subsequent to the formation of the thin hatching envelope, the ring vesicles undergo exocytosis resulting in an accumulation of ring-shaped structures in the perivitelline space. These structures coalesce and form an electron translucent layer on the inner surface of the thin elevated envelope to form the thickened hatching envelope. The formation of the cortical vesicles, their exocytosis and the elaboration of the hatching envelope are normally completed within 40-45 min after spawning.

The rapid electrical depolarization of the egg's plasma membrane which protects sea urchin ova against polyspermy in the interval between stimulation by the fertilizing spermatozoon and completion of the cortical reaction is believed to be mediated by the influx of sodium (Na+) ions. This hypothesis was tested in Arbacia punctulata and Strongylocentrotus purpuratus by inhibiting the rapid block to polyspermy with low-Na+ (choline-substituted) seawater, and the cortical granule secretion-mediated block with soybean trypsin inhibitor (SBTI). Eggs inseminated in low-Na+ seawater or SBTI became heavily polyspermic. Polyspermy elicited by low Na+ or SBTI was increased in dejellied Strongylocentrotus eggs. However, the severity of polyspermy was not enhanced in low Na+ plus SBTI because the fertilizing capacity of sperm and gamete binding were reduced in low-Na+ media. Since SBTI completely suppresses the cortical granule secretion-mediated block to polyspermy in Arbacia for about 3 min postinsemination, the rate at which SBTI-treated eggs became polyspermic was used to measure the duration and efficacy of the rapid block. The half-time for SBTI-treated Arbacia eggs to become polyspermic in natural (425 mM Na+) seawater was 89.9 ± 4.7 sec (N = 4). The plot of incidence of polyspermy vs time was essentially an inverse mirror image of electrophysiologic data on repolarization of the oolemma during fertilization. The rapid block is also Na+ dependent, since SBTI-treated eggs became polyspermic more rapidly in 26 mM Na+ seawater (half-time, 15.8 ± 1.6 sec; N = 3, P < 0.01).

The fine structure of the zona pellucida of mouse unfertilized egg cells and of mouse embryos was investigated. In addition to standard fixation for electron microscopy, fixation with an addition of ruthenium red was used and found to be especially valuable for the purpose of studying the zona pellucida. The zona pellucida of unfertilized egg cells and of embryos consisted mainly of fibrillar and granular material. There were two layers: a thicker internal and a denser external layer in the zona pellucida of the unfertilized egg cells. The zona pellucida of embryos had, in addition to these two layers, a coarsegrained intimal layer inside the internal layer and a fine-grained peripheral layer outside the external layer. The intimal layer seems to be the product of cortical reaction whereas the peripheral layer can be accumulated and modified during the passage of the embryo through the female genital tract. The influence of developmental events and environmental factors on the fine structure of zona pellucida was discussed.

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (TmPase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg. Our results are consistent with the view that membrane, retrieved from the egg surface during the cortical reaction and shuttled into organelles with lysosomal properties (i.e., secondary lysosomes and multivesicular bodies), undergoes degradation. However, our study does not exclude other possibilities for handling internalized membrane, including storage and recycling to the plasma membrane.

The egg coats of a starfish (Patiria miniata) are examined before, during, and after the cortical reaction by scanning and transmission electron microscopy. The unfertilized egg is closely invested by a vitelline coat about 300 micron thick, and cortical granules are scattered in the peripheral cytoplasm. After insemination, as the cortical granules undergo exocytosis, the cortical reaction sweeps over the egg surface. Much of the material ejected from the cortical granules adheres to the inner surface of the vitelline coat as a dense layer about 40 micron thick and as scattered spheres and hemispheres, each about 1 micron in diameter. Together, the vitelline coat and the adherent cortical granule material form the fertilization envelope, which becomes separated from the plasma membrane of the egg by a perivitelline space. The perivitelline space contains some flocculent material, which is too diffuse and discontinuous to be considered a hyaline layer. Possible functions of the starfish egg coats are discussed.

Ion requirements for exocytotic release and assembly of secretory products have been examined in surface complexes isolated from Arbacia punctulata eggs. Ca2+ and other components of artificial seawater, contained in simplified media, have been shown to modulate the cortical reaction in vitro. The ultrastructural appearance of the fertilization envelopes and the “mosaic” membrane sheets, formed during exocytosis, were varied as a function of divalent ion concentration. Moreover, both Ca2+ and Na+ were required for envelope assembly. In the presence or absence of ATP, higher levels of Ca2+ were required for exocytotic release than reported for other species. Further, Mg2+ was found to inhibit Ca2+ activation of secretion when K+ was omitted from the incubation mixture containing the surface complex. Together these data indicate that structural modification of the egg surface during the cortical reaction is primarily dependent on the extracellular milieu.

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.

A consistent response of mammalian ova to penetration by sperm is the total or nearly total loss of cortical granules; this has been designated the cortical reaction. The phenomenon appears important in preventing polyspermic fertilization. This cortical reaction is not a constant feature of parthenogenetic activation of mammalizn ova. Ova activated by other methods retain cortical granules or lose only some of them. The occurrence of the cortical reaction in mouse ova activated by electric shock in vivo is reported. Swiss mice were induced to ovulate by ip injection of pregnant mares serum gonadotropin followed after 44-48 hours by 5 IU of human chorionic gonadtoropin. 2-3 1/2 hours after assumed ovulation electric stimulation of 40 volts was applied to 1 ampulla. The contralateral ampulla served as a control. 1-8 hours after electric shock ova were fixed in situ or flushed from the oviducts and prepared for electron microscopy. Of 89 ova recovered from stimulated oviducts 36 were found to be in various stages of activation with completion of the 2nd stage of meiotic division. Controls and nonactivated ova all showed numerous cortical granules. The cortical or cortical bus cytoplasm of control ova also contained prominent Golgi complexes apparently involved in the synthesis of cortical granules. Of the activated ova 33 of 36 showed total or nearly total absence of cortical granules and absence or atrophy of Golgi complexes. Under favorable conditions ova activated by electric shock in vivo can attain development only up to the 8-somite stage.

Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.

In 1902, Boveri introduced the important concept that for the success of fertilization the gametes must activate one another. Based primarily on studies on the sea urchin and ascidian fertilization the suggestion is presented here that “activation” of the spermatozoon actually involves a switching off of its metabolic machinery as a result of its interaction with the sperm receptors of the egg envelopes and prior to its fusion with the egg.

The eggs of most mammals at ovulation are metabolically relatively inert cells arrested in metaphase II of meiosis. In response to an activation stimulus normally provided by the fertilizing sperm but induced artificially by a number of parthenogenetic agents, the egg resumes meiosis, undergoes a cortical reaction, and becomes metabolically more active. Visible evidence for the resumption of meiosis involves the abstriction of a second polar body at approximately 30 min postactivation. In the cortical reaction, the egg undergoes the exocytotic release of its cortical granules, a process that results in formation of a new mosaic plasma membrane from the fusion of limiting cortical granule membranes with the egg plasma membrane. At the same time, cortical granule contents released into the perivitelline space come in contact with the egg plasma membrane and the zona pellucida. The cortical reaction and granule exocytosis are of primary interest to any discussion of polyspermy for cortical granule contents have been associated with polyspermy-preventing mechanisms in the eggs of animals of diverse species (for review, see Schuel, 1978, or Gulyas, 1980).