Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2, and ZP3), of which ZP2 is proteolytically cleaved after gamete fusion to prevent polyspermy. This cleavage is associated with exocytosis of cortical granules that are peripherally located subcellular organelles unique to ovulated eggs. Based on the cleavage site of ZP2, ovastacin was selected as a candidate protease. Encoded by the single-copy Astl gene, ovastacin is an oocyte-specific member of the astacin family of metalloendoproteases. Using specific antiserum, ovastacin was detected in cortical granules before, but not after, fertilization. Recombinant ovastacin cleaved ZP2 in native zonae pellucidae, documenting that ZP2 was a direct substrate of this metalloendoprotease. Female mice lacking ovastacin did not cleave ZP2 after fertilization, and mouse sperm bound as well to Astl-null two-cell embryos as they did to normal eggs. Ovastacin is a pioneer component of mouse cortical granules and plays a definitive role in the postfertilization block to sperm binding that ensures monospermic fertilization and successful development.     

An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata

Fine structural studies have been conducted to determine the role of the trypsinlike proteinase during fertilization which is localized within the cortical granules of sea urchin eggs. Eggs were treated with soybean trypsin inhibitor (SBTI), inseminated, and prepared for microscopic examination at various intervals during fertilization. In the presence of SBTI the breakdown of the cortical granules is delayed and the elevation of the vitelline layer is incomplete. Although all the cortical granules in SBTI-treated eggs are dehisced in a manner similar to controls, the rate at which their breakdown is propagated is reduced. Consequently, dehiscence of all the cortical granules in SBTI-treated specimens requires approximately 3 min to complete, vs. 1 min for controls. In addition the fertilization membrane has a blistered appearance owing to its attachment, at multiple loci, to the surface of the zygote. During the early stages of fertilization many of the treated eggs are monospermic, later all are polyspermic. Supernumerary sperm enter treated eggs (zygotes) up to 10 min after the initiation of the cortical granule reaction, apparently at those sites where the fertilization membrane has failed to separate from the zygote's surface.     

Fertilization in a chiton: Acrosome-mediated sperm-egg fusion

Contrary to the widely accepted view that chiton sperm lack acrosomes and that fertilization in this group occurs via a micropyle, we demonstrate here that fertilization in Tonicella lineata occurs by acrosome-mediated sperm-egg fusion. The acrosome is a small vesicle containing two granules located at the tip of the sperm. The eggs have an elaborate hull (=chorion), which is formed into cupules that remain covered by follicle cells until maturity. When dissected ripe eggs were exposed to sperm in vitro, the sperm were attracted only to open cupules, inside which they swam through one of seven channels to the base where they penetrated the hull. The acrosome fired on contact with, or in, the hull, and during passage through it the apical granule was exhausted while the basal granule was exposed. If sperm contacted follicle cells between the cupules the acrosome did not react. The vitelline layer beneath the hull contains pores arranged in a regular pattern. Embedded in the base of each pore is an egg microvillus. Having penetrated the hull the sperm anterior filament located a pore and fused with the tip of the egg microvillus projecting into it. This created a membranous tube, through which the sperm nucleus was injected into the egg. The egg membrane appeared to be raised up into a small fertilization cone around the penetrating sperm, the vitelline layer became slightly elevated, and some cortical granules were released by exocytosis.     

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N- acetylglucosaminidase found in cortical granules was identified as beta- hexosaminidase B, the beta, beta homodimer. Inhibition of N- acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase- binding site, accounting for the block in sperm binding to the zona pellucida.     

Role of specialized microvilli and the fertilization envelope in the spatial positioning of blastomeres in early development of embryos of the starfish Astropecten scoparius

In the eggs of a wide range of animal species, various factors that determine the blastomeres' presumptive fate are known to locate unevenly within the egg. In the embryos of these animals, cleavage occurs not just to increase cell numbers, but also to distribute the factors to the respective blastomeres, resulting in cell specialization at the later stages. In the early cleavage stages, before the establishment of a device such as desmosomes to directly join the blastomeres, some other means is needed to keep the blastomeres together and maintain the relative positions among them. In this study, we found that the embryos of the starfish Astropecten scoparius lack the hyaline layer seen in sea urchin embryos and that blastomeres adhere to the fertilization envelope (FE) via filamentous cellular projections (fixing processes). Electron microscopy revealed the fixing processes to be specialized microvilli formed, after the elevation of the FE, by the elongation of short microvilli that pre-exist in unfertilized eggs. After the first cleavage, the two blastomeres separate from each other and finally attach to the FE. In the subsequent cleavages, the blastomeres undergo repeated cell division without separating from the FE. Between the blastomeres and the FE, only shortened fixing processes were observed. Destruction of the fixing processes caused release of the blastomeres from the FE and disturbance of the relative positions of the blastomeres, resulting in abnormal development of the embryos. These observations suggest that the fixing process is a device to keep the egg placed centrally in the FE up to the first cleavage, and after the first cleavage and beyond to anchor the blastomeres to the FE so that the FE can be used as a scaffold for morphogenesis. Electron microscopy also suggests that the inner layer of the FE, which is derived from the contents of cortical granules, reinforces the adhesion of the fixing processes to the FE. Immuno-electron microscopy, using an antibody against sea urchin hyaline layer, showed that the inner layer of the FE of starfish eggs and the hyaline layer of sea urchin eggs, which are both derived from cortical granules, contain some common elements.     

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.     

Cortical granule exocytosis in hamster eggs requires microfilaments

Earlier work has demonstrated that hamster eggs that do not release a second polar body after fertilization in vitro lack a block to polyspermy (Stewart-Savage and Bavister, 1987: Gamete Res 18:333–338). Since polar body release requires microfilaments, the involvement of microfilaments in cortical granule exocytosis was examined. When hamster eggs were treated with cytochalsin B (CB) for 1 hr and then coincubated with sperm for 90 min, there was a dose-dependent increase in both the percentage of eggs with more than one sperm penetrating the zona pellucida and the mean number of sperm that penetrated the zona, with a maximum effect at 20 μg CB/ml (100% polypenetration, 3.0 ± 0.3 sperm/egg). Cytochalasin-treated eggs retained 85% of their cortical granules 55 min after insemination, as compared to unfertilized eggs. Longer time periods did not result in any further reduction. As seen with the scanning confocal microscope, an extensive microfilament network was present in the cortex of untreated eggs, with the cortical granules located within this cortical network. The cortical microfilament network was highly reduced in CB-treated eggs. When viewed with the electron microscope, the same number of cortical granules were located next to the plasma membrane in both cytochalasin-treated and untreated, unfertilized eggs. These data indicate that intact microfilaments are required for normal cortical granule exocytosis in the hamster egg, but the role of the microfilaments in exocytosis is unresolved. Mol. Reprod. Dev. 47:334–340, 1997. © 1997 Wiley-Liss, Inc.     

The vitellin-processing protease of Blattella germanica is derived from a pro-protease of maternal origin

Proteolytic processing of vitellin in Blattella germanica embryos is accomplished by activation of a yolk-borne cysteine protease (Mr 29 000) derived from a pro-protease precursor of Mr 40 000 (Liu et al., 1997). In the present study, fat body, ovaries and embryos of different developmental stages were examined immuno-cytochemically with purified murine anti-proprotease antibodies (Liu, 1995) to determine the intracellular location of the pro-protease. Proenzyme was detected in discrete secretory granules of the fat body and in large lysosome-like vesicles of both the follicle cell cytoplasm and the cortical ooplasm of previtellogenic ovarian follicles. In vitellogenic oocytes, coated pits and vesicles are scantily labelled for proprotease and no clear gold pattern could be discerned over the yolk granules. During embryonic development, pro-protease is associated with some, but not all, yolk granules. In newlyovulated eggs (day 0), pro-protease is either distributed over the entire granule or confined to some internal vesicles. As development proceeds, it becomes associated with almost every yolk granule and restricted to the superficial layer. By day 6, pro-protease is evident over all yolk granules but the intensity of reaction has greatly diminished, due probably to conversion of the pro-protease to the mature enzyme. Yolk granules are flanked along their margin by vesicles that are stained after zinc-osmium fixation. This observation suggests that the pro-protease may be transferred between yolk granules via vesicular shuttling. B. germanica embryos of different developmental stages were also exposed to [(3)H]-DAMP. Data show that autoradiographic grains are not evenly distributed among closely adjacent yolk granules within vitellophagic cells, a result consistent with the known slight temporal asynchrony of the acidification event.     

Surface ultrastructure of paddlefish eggs before and after fertilization

The surface ultrastructure of eggs of the paddlefish Polyodon spathula was investigated by scanning electron microscopy. Mature eggs of paddlefish possess four to 12 micropyles in the animal polar region. There are sperm entry sites in the egg surface under the micropyles which consist of tufts of microvilli. Five to nine sperm entry sites were observed on mature eggs. Probably, the number of sperm entry sites corresponds to the number of micropyles. In a few eggs, 1 min after fertilization the ball-like enlarged top of a cytoplasmic process (probably a full-grown fertilization cone) had reached the external aperture or the canal of several micropyles. In other micropyles of the same egg, a few smaller cytoplasmic processes or flocculent material were found in the micropylar canal. With one exception, no sperm tails were found there. The formation of the full-grown cytoplasmic process is possibly initiated before the cortical reaction has started in an area of the animal hemisphere. Three, 10 and 20 min after fertilization, the uneven surface of the cortical cytoplasm in the animal polar region rose gently where microvilli were much less than the in other area and together with a secondary polar body at the latter stage. Taken together, paddlefish eggs may have sperm entry sites corresponding to the number of micropyles and respond to the stimulus of fertilization by forming a few cytoplasmic processes–fertilization cones (larger and smaller). Sperm penetration into the egg may be achieved at an earlier stage of fertilization (sperm-egg contact), as inferred from the fact that a secondary polar body was formed at the 20-min stage irrespective of the exceptional finding of the sperm tail.     

Time course and pattern of cortical granule breakdown in hamster eggs after sperm fusion.

We have determined the temporal relationship between sperm fusion and cortical granule breakdown in the hamster egg. Sperm fusion was determined by the Hoechst-transfer method (Stewart-Savage and Bavister: Dev Biol 128:150-157, 1988), and cortical granules were visualized with fluorescein isothiocynate-conjugated Lens culinaris agglutinin (Cherr et al. J Exp Zool 246:81-93, 1988). By 55 min after insemination, there was an 85% reduction in the density of cortical granules (fewer than four granules/100 microns2). Taking this value as the completion of the cortical reaction, analysis of the data indicate that the cortical reaction was completed 9 min after sperm fusion and 3 min after the formation of the zona and cell surface blocks to polyspermy. There was no obvious spatial pattern of granule loss in eggs that had a Hoechst-positive sperm but had not completed the cortical reaction.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Subnormal zona pellucida change in parthenogenetic mouse embryos

Parthenogenetic mouse embryos were obtained by the method of electrical stimulation of eggs in vivo(Tarkowski et al., 1970), and their developmental retardation and limited viability were confirmed. Very early deviations from normalcy seemed likely in these embryos, and we chose to investigate their “zona reaction,” as this is one of the earliest events identified (Braden, et al., 1954) in normal fertilization. The change, ordinarily triggered by sperm penetration of the egg, decreases the permeability of the zona pellucida to supernumerary sperms, and has been attributed (Austin and Braden, 1956) to products released by cortical granules.An indirect assay for the state of the zona pellucida is presented. It is based on the observation (Mintz, 1970) that pronase, other proteolytic enzymes, and the normal uterine zonalytic factor lyse zonas of fertilized eggs more slowly than those of unfertilized eggs. Comparative zona lysis times in pronase are thus employed as a test for the degree of zona change after parthenogenetic activation relative to that after activation by sperm.The zonas of parthenogenetic embryos in stages ranging from 2 to 14 cells varied in their lysis times in pronase and overlapped with those of unfertilized and fertilized egg zonas. As a population, the zonas of the parthenogenones had intermediate lysis times. Thus, in the strains tested, electrical shock evokes only a partial zona reaction and, in this respect, is not an adequate substitute for sperm penetration.A working hypothesis for future testing is that the subnormal zona change and retarded development may both be due to inadequate release of products from cortical granules, under these conditions of artificial activation of the mouse egg.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

A simplified method of generating transgenic Xenopus

Currently transgenic frog embryos are generated using restriction-enzyme-mediated integration (REMI) on decondensed sperm nuclei followed by nuclear transplantation into unfertilized eggs. We have developed a simplified version of this protocol that has the potential to increase the numbers of normally developing transgenic embryos.     

Premature cortical granule loss does not prevent sperm penetration of mouse eggs.

The role of cortical granules in the mouse egg's plasmalemma block to polyspermy was investigated by examining the effect of premature granule loss on egg fertility. Granule loss, quantitated by transmission electron microscopic examination, was induced in zona-free eggs by exposure to the divalent cation ionophore, A23187, or by mechanical removal of zonae. Egg exposure to ionophore led to the loss of approximately 50% of the egg's complement of granules in the absence of nuclear activation (parthenogenesis), while complete cortical granule loss accompanied the parthenogenetic activation seen in a limited population of mechanically stimulated eggs. Aged eggs underwent nuclear activation without a dramatic reduction in granule complements. The fertility of treated zona-free eggs was identical to that of controls, as measured by the percentage of eggs penetrated and by the mean number of sperm recovered per egg. Moreover, both ionophore-treated and aged eggs subsequently underwent a normal sperm-induced block response. Exposure of zona-intact eggs to ionophore was also without effect on egg fertility. These results indicate that cortical granules are not involved in the plasmalemma block to polyspermy in the mouse.     

Localization of cortical granule constituents before and after exocytosis in the hamster egg

Electrical activation of the hamster egg was used to study cortical granule constituents before and after exocytosis. The activated hamster eggs underwent cortical granule decondensation just prior to and at the time of exocytosis. Some of the cortical granules of aged, unactivated eggs underwent similar changes. FITC- and gold-conjugated Lens culinaris agglutinin (LCA) bound intensely to the surfaces of activated but not unactivated eggs. This labelling was associated with the microvilli. Permeabilized eggs exhibited discrete cortical labelling before activation, with a subsequent decrease following the cortical reaction. Gold-conjugated LCA specifically bound to cortical granules when incubated with thin sections. FITC-soybean trypsin inhibitor (SBTI) bound in discrete foci in the cortex of unactivated eggs. Following activation, cortical labelling by SBTI decreased. Aprotinin and benzamidine hydrochloride inhibited FITC-SBTI from binding to the egg cortex. Gold-avidin localization of biotin-SBTI in the electron microscope demonstrated that condensed cortical granules did not bind SBTI but decondensed or exocytosing granules did. This suggests that a cortical granule protease is exposed just prior to exocytosis. Activated eggs exhibited dramatic decreases in the number of hamster sperm penetrating the cytoplasm, suggesting that a plasma membrane block to polyspermy is temporally related to cortical granule exocytosis.     

Formation and structure of the fertilization envelope in Xenopus laevis

This paper reports the morphological events that occur when the vitelline envelope (VE) of an unfertilized egg of Xenopus laevis is transformed into the fertilization envelope (FE) surrounding the zygote. The VE is about 1 μm thick and is composed of an interlacing network of small filaments. The FE is constructed from the VE plus an electron-dense layer (fertilization layer), about 2–6 μm thick, on the outer surface of the VE, i.e., at the interface between the VE and the innermost jelly-coat layer. The fertilization layer is a stable component of the FE and is not removed by mercaptan solutions used to dejelly eggs. The events of FE formation were observed in the light and electron microscopes after dejellied eggs were activated by pricking. The FE is established when material from the cortical granules is extruded into the perivitelline space. The cortical granule material passes through the VE as the envelope lifts away from the egg surface. Some cortical granule material deposits in the interstices of the VE, but most of it forms the fertilization layer on the outer surface of the envelope. The cortical reaction is completed about 8–9 min after addition of sperm when eggs are fertilized in vitro.     

OCCURRENCE AND FINE STRUCTURE OF SUBCORTICAL CYTOPLASMIC ISLETS IN FERTILIZED EGGS OF CYNOPS PYRRHOGASTER

Fertilized and unfertilized eggs of Cynops pyrrhogaster were examined by light and electron microscopy. In fertilized eggs that have just been laid, there are numerous small cytoplasmic patches free of granules in the pigmented layer of the animal hemisphere. Many of these granule-free cytoplasmic islets gradually grow out subcortically from the pigmented layer and fuse to form a subcortical layer of yolk-free cytoplasm of varying thickness by the time of the first cleavage division. The cytoplasmic islets are present in 100% of the fertilized eggs, but not in unfertilized eggs. Electron microscopic observations showed that the cytoplasmic islets contain tubules and that development of a complex system of cortical tubules constitutes the basis of the early growth of the cytoplasmic islets. The cortical tubules are transient structures and are no longer observable a few hours after the eggs are laid. These phenomena are considered to be a response of the egg to the fertilization stimulus.     

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

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

In vitro activation of Drosophila eggs

Mature ovarian eggs of Drosophila can be activated by treatment with hypotonic buffers. Well-fed, 4-day-old virgin flies contain large numbers of partially dehydrated mature eggs. When these eggs are transferred to a hypotonic culture medium, the ovarian eggs swell immediately and within minutes up to 70% become impermeable. The following cellular events ensue: meiosis, which had been arrested at metaphase I, is completed and the "polar body" nuclei fuse; cortical multivesicular bodies with acid phosphatase activity appear within minutes; polar granules fragment, dissociate from mitochondria, and become associated with polysomes; finally, monosomal ribosomes move to the polysomal region of a sucrose gradient. Each of these events corresponds to the normal in vivo effects of ovulation of oocytes, whether or not they are fertilized. When ovarian eggs of a parthenogenetic strain of D. mercatorum were activated by hypotonic treatment, some eggs developed into normal embryos. The presence of high potassium, low pH, and polyethylene glycol enhanced the frequency of normal development. Thus, we suggest that the rehydration of mature oocytes, as they move from the ovary to the uterus, activates the maternal program of the oocyte.