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At fertilization in mammals, the spermatozoon triggers a unique signal transduction mechanism within the egg, leading to its activation. It is well accepted that the earliest event observed in all activated eggs is an abrupt rise in intracellular calcium concentrations. However, little is known regarding the downstream proteins that are activated by this rise in calcium. Calpains constitute a family of intracellular calcium-dependent cysteine proteases whose members are expressed widely in a variety of cells. We investigated the expression and possible role of the calpain isoforms μ and m throughout egg activation. Both calpains were expressed in the rat egg and localized at the egg cortex as well as in the meiotic spindle. m Calpain translocated to the membrane and to the spindle area during parthenogenetic egg activation and duringin vivofertilization, upon sperm binding to the egg. The cytoskeletal protein α-spectrin (fodrin) was proteolysed by calpain during the egg-activation process, as demonstrated by specific calpain-breakdown products. Following parthenogenetic activation by ionomycin or puromycin, the calpain-selective permeable inhibitor, calpeptin, inhibited the resumption of meiosis and cortical reaction in a dosedependent manner. Calpeptin was also effective in inhibitingin vitrofertilization. These results may imply a correlation between calpain activation and mammalian egg activation at fertilization and a possible role for calpain in the cascade of cellular events leading to resumption of meiosis.

Following fusion of sperm and egg, the contents of cortical granules (CG), a kind of special organelle in the egg, release into the perivitelline space (cortical reaction), causing the zona pellucida to become refractory to sperm binding and penetration (zona reaction). Accumulating evidence demonstrates that mammalian cortical reaction is probably mediated by activation of the inositol phosphate (PIP2) cascade. The sperm-egg fusion, mediated by GTP-binding protein (G-protein), may elicit the generation of two second messengers, inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). The former induces Ca2+ release from intracellular stores and the latter activates protein kinase C (PKC), leading to CG exocytosis. Calmodulin-dependent kinase II (CaMKII) may act as a switch in the transduction of the calcium signal. The CG exudates cause zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Oolemma modification after sperm-egg fusion and formation of CG envelope following cortical reaction also contribute to polyspermy block. Microsc. Res. Tech. 61:342–348, 2003. © 2003 Wiley-Liss, Inc.

Physiological events at the time of fertilization of pig oocytes may differ in vitro depending on the in vitro fertilization (IVF) medium. This hypothesis was tested by in vitro maturation of pig oocytes for 44 h in NCSU-37 medium and thereafter fertilization with frozen-thawed ejaculated spermatozoa. Three different IVF media (TCM-199, Tyrode's albumin lactate pyruvate (TALP) and Tris-buffered medium (TBM)) were used. For the acrosome reaction test, spermatozoa were incubated for 0-150 min in the three IVF media, and the proportion of live acrosome-reacted and acrosome-intact cells was determined by fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA) and propidium iodide (PI) staining. The cortical granule density of oocytes was evaluated by confocal microscopy, 2.5 and 5.0 h after culture in each medium in the presence or absence of spermatozoa. Zona pellucida resistance to pronase digestion was also determined in the same groups. The percentages of penetration, monospermy, male pronucleus formation, cleavage and blastocyst formation, and the number of cells per blastocyst after culture were determined. The results indicate that the acrosome reaction occurred much faster in TBM than in TCM-199 or TALP medium. Continuous cortical granule synthesis was observed in the three media when oocytes were incubated in the absence of spermatozoa. The presence of spermatozoa triggered the cortical reaction in a large proportion of oocytes fertilized in TCM-199 and TALP media. On the basis of the duration of pronase digestion, the zona pellucida of oocytes incubated in TCM-199 was harder (407.7 +/- 35.5 s) than that of oocytes cultured in TALP (235.4 +/- 18.2 s) or TBM (189.1 +/- 16.8 s). No zona pellucida hardening was noted in oocytes after insemination in any of the media. The percentages of penetration and cleavage were higher in oocytes cultured in TCM-199 and TALP than in TBM. The percentage of monospermy was higher in TCM-199 and TBM than in TALP. No effect of the medium was shown on the percentage of blastocyst formation or on the number of cells per blastocyst. In conclusion, the results highlight how differently the fertilization events take place in each IVF medium and how far these IVF media still are from achieving biological properties of gametes close to those observed in the physiological setting.

Publisher Summary This chapter explores the membrane events of egg activation involving a number of morphological and biochemical changes like the raising of the fertilization envelope in the sea urchin. The plasma membrane of the egg undergoes fusion with the sperm to initiate the process of fertilization, and the endoplasmic reticulum inside the egg undergoes a concerted release of Ca 2+ ions that are now known to be the essential trigger for the development of the egg into an embryo. Even though the membrane's potential change may not play a direct role in egg activation in sea urchins, studies of the electrophysiology of fertilization in this species have provided some important data for theories of the activation of an egg by the sperm. Increases in the plasma membrane electrical capacitance can be measured at fertilization. The main capacitance increase is due to the addition of membrane surface area caused by the exocytosis of cortical granules. The latent period of fertilization has been a term used in the sea urchin field to indicate the delay between the initial sperm-egg interaction and the initiation of the cortical reaction. The data on the timing of the latent period are not as extensive in any species outside the sea urchin. Despite many years of research effort, there have been few sperm-derived molecules that can actually be shown to trigger the correct pattern of Ca 2+ changes in appropriate eggs. In some species, sperm-derived molecules have been applied to the surface and shown to activate eggs.

Denuded Bufo arenarum oocytes matured in vitro by progesterone treatment exhibited abnormal segmentation due to the penetration of more than one sperm. These oocytes were able to respond to activation stimuli and exhibited the external signs characteristic of activation. However, the prevention of polyspermy was not effective in these oocytes, which exhibited numerous sperm in their cytoplasm. The aim of this work was to analyse the cortical reaction in polyspermic Bufo arenarum oocytes matured in vitro. The result indicate that the cortical reaction of these oocytes seems to occur with a chronological sequence similar to that described for ovoposited oocytes of this species. In addition, when, 1 min after pricking, cortical granule exocytosis occurred, the oocytes became refractory to sperm entry, suggesting that they are able to establish a slow block to polyspermy.

Cysteine string proteins (csps) are associated with secretory organelles in a wide range of eukaryotic cells. Functional studies of these proteins indicate that they subserve one or more vital steps in the pathway of regulated exocytosis. Here, we document the presence of csps in fully grown (stage VI) oocytes of the frog, Xenopus laevis. Both Northern and immunoblot data support the conclusion that csps are expressed in these cells. In addition, immunoreactive csp is seen even at the earliest stage of oocyte development, namely, in stage I oocytes. Finally, immunoblot and immunocytochemical results indicate that csps are associated with cortical granules of stage II–VI oocytes. These observations suggest that csps participate in the cortical reaction that underlies the sustained block to polyspermy in Xenopus eggs. Moreover, because of the relative ease of manipulating cells as large as Xenopus oocytes, this system harbors considerable promise as a model for studying the role of csps and other proteins in exocytotic events.

Orientation-selective neurons in monkeys and cats show contrast saturation and contrast-invariant orientation tuning (Albrecht and Hamilton, 1982). Recently proposed models for orientation selectivity predict contrast invariant orientation tuning but no contrast saturation at high strength of recurrent intracortical coupling, whereas at lower coupling strengths the contrast response saturates but the tuning widths are contrast dependent (Hansel and Sompolinsky, 1997; Bartsch, Stetter and Obermayer, 1997). In the present work we address the question, if and under which conditions the incorporation of a stochastic distribution of activation thresholds of cortical neurons leads to the saturation of the contrast response curve as a network effect. We find that contrast saturation occurs naturally if two different classes of inhibitory inter-neurons are combined. Low threshold inhibition keeps the gain of the cortical amplification finite, whereas high threshold inhibition causes contrast saturation.

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Machaty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 microM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 +/- 1.14, was observed 509.64 +/- 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 +/- 11.7 CGs/100 micron 2 of cortex in control oocytes to 25.7 +/- 19.2 CGs/100 micron 2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 +/- 10.4 CGs/100 micron 2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 +/- 5.1 CGs/100 micron 2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 +/- 14.8 CGs/100 micron 2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.

Cytological and cytochemical studies were carried out to investigate the surface characteristics of oocytes of four teleost species from the Sao Francisco river. The fishes were submitted to hypophysation at the Tres Marias Hybrobiology and Fishculture Station, Minas Gerais, Brazil, in January 1996. Postspawning, oocytes of the curimatas Prochilodus affinis, Prochilodus marggravii and dourado Salminus brasiliensis were surrounded by a thick, three-layered zona pellucida with radial striae. The surface of spawned oocytes of the surubim, Pseudoplatystoma coruscans, was comprised of mucous coat located externally to a thin, two-layered and striated zona pellucida. Oocyte activation during fertilization, lead to cortical reaction, formation of a perivitelline space, reduction of the thickness of the zona pellucida and increase in the oocyte diameter in the four species. Following fertilization, many spermatozoa were embedded in the mucous coat of the surubim oocytes. During embryogenesis, this later coating became thicker, diffuse and less viscous while the zona pellucida (chorion) was thinner in all studied species. Cytochemical analyses indicated species-specific differences in the oocyte surface after spawning. It was suggested that the mucous coat of surubim oocytes play a functional role during fertilization. The knowledge of the morphology of the oocyte surface of teleost is important for our understanding of the interactions between their eggs and surrounding environment and may also contribute significantly to phylogenetic studies.

The cortical reaction (CR) in mammalian oocytes is induced following sperm‐egg membrane fusion. During intracytoplasmic sperm injection (ICSI) the physiological cascade of gamete interaction events is bypassed. The aim of this study was to explore CR occurrence after the ICSI procedure and its correlation with the meiotic status of the oocytes. Unfertilized and abnormally fertilized (one- or three-pronucleate) human oocytes were investigated. The chromosomal status was analysed by Hoechst staining. The CR occurrence and its fluorescent pattern were assessed by confocal scanning laser microscope using the lectin lens culinaris and Texas Red staining. Our results reveal a positive correlation between the activation of oocytes and their CR. No CR was demonstrated in unfertilized-unactivated oocytes with or without sperm in their cytoplasm. In partially activated oocytes showing resumption of meiosis but no formation of pronuclei, a moderate CR was observed. In fully activated oocytes displaying one or three pronuclei, a strong CR was monitored. It was concluded that in ICSI, as in physiological fertilization, oocyte activation is a prerequisite for CR.

The zona pellucida is the extracellular coat that surrounds the mammalian oocyte. It forms a spherical shell of remarkably uniform thickness (5-10 microns in eutherian mammals). The mouse is currently the largest source of data on the zona pellucida and this review is built largely on these data. The zona pellucida is composed of three proteins in both mice and humans: ZP1, ZP2 and ZP3. These proteins are glycosylated and, in mice, have mature relative molecular masses of 200,000, 120,000 and 83,000, respectively. ZP1 is a dimer of two apparently identical subunits. All three mouse proteins have been sequenced and possess transmembrane domains at their C-terminal ends coupled with furin cleavage sites immediately upstream. Sequence data have been used to provide an accurate assessment of the mole ratios of the three proteins. The ratio of ZP2:ZP3 is close to 1:1, whereas ZP1 is approximately 9% of the combined mole amounts of ZP2 and ZP3. Ultrastructural evidence suggests that the mouse zona pellucida is composed of filaments constructed by head-to-tail association of globular proteins. The coordinate synthesis of the three zona pellucida proteins coupled with the near 1:1 stoichiometry of ZP2 and ZP3 is consistent with a model in which ZP2-ZP3 heterodimers are the basic repeating units of the filament, with cross-linking of filaments by dimeric ZP1. This model is also consistent with data from ZP2 and ZP3 gene knockout and antisense experiments. However, the structure remains unproven. The small amount of ZP1 relative to ZP2 and ZP3 may have important implications for the distribution of ZP1 cross-links, since the number of cross-linking sites potentially exceeds the number of ZP1 dimer molecules by a considerable margin. The evidence that ZP1, ZP2 and ZP3 are all synthesized via a membrane-bound step is discussed and two models are proposed for the assembly of the zona pellucida. The cortical reaction and its effect on the zona pellucida are examined in detail. It is shown that the amount of material released by cortical granules could be of the order of 30% by mass of ZP1, and that if this material was distributed predominantly on the inner face of the zona pellucida, its local concentration could approach that of ZP1. A model in which the zona block to polyspermy is caused by direct titration of zona pellucida binding sites is suggested as an alternative to the explanation that relies on enzyme cleavage of ZP2 to ZP2f. Finally, some of the major experimental and structural issues that remain to be addressed are identified.

Few embryos derived from pig oocytes matured and inseminated in vitro are able to develop to blastocyts in culture. The present study was conducted to examine the effects of oocyte maturation media on the developmental ability of pig oocytes matured and inseminated in vitro. Follicular oocytes collected from ovaries of prepubertal gilts were cultured in NCSU23 medium, tissue culture medium 199 or a modified Whitten's medium. All of the media were supplemented with 0.57 mmol cysteine l-1 and 10% pig follicular fluid. After maturation, some of the oocytes were used for examination of intracellular glutathione content, nuclear maturation and cortical granule distribution. The other oocytes were inseminated in vitro in a modified Tris-buffered medium with cryopreserved, ejaculated spermatozoa for examination of cortical reaction, sperm penetration, male pronuclear formation and blastocyst development. No differences (P > 0.05) were observed in nuclear maturation, cortical granule distribution, sperm penetration, male pronuclear formation, polyspermy and cleavage in oocytes matured in the three media. However, significant (P < 0.05) differences were observed in glutathione content, cortical granule exocytosis, blastocyst development and number of cells in blastocysts. NCSU23 medium gave the best results of the three media, resulting in 5.8 pmol glutathione per oocyte, 97% of cortical granule exocytosis, 30% blastocyst development and 36.8 +/- 17.0 cells per blastocyst. These results clearly indicate that cytoplasmic maturation of pig oocytes was significantly affected by oocyte maturation media even in the presence of cysteine and pig follicular fluid. In addition, it was demonstrated that a large proportion of pig oocytes can develop to blastocysts under in vitro conditions.

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca2+ in cells ([Ca2+]i). Ca2+ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984). The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca2+- and calmodulin-dependent protein phosphatase, Ca2+-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca2+- and calmodulin control of sperm motility in sea urchins.

The cortical reaction induces changes at the egg's Zona pellucida (ZP), perivitelline space and/or oolemma level, blocking polyspermic fertilization. We studied the timing of sperm penetration and cortical reaction in pig oocytes matured under different conditions and inseminated with different boars. Immature (germinal vesicle stage) and in vitro matured (IVM) (metaphase II stage) oocytes were inseminated and results assessed at different hours post insemination. Penetrability and polyspermy rates increased with gamete coincubation time and were higher in IVM oocytes. A strong boar effect was observed in IVF results. Cortical reaction (assessed as area occupied by cortical granules) and galactose-β(1-3)-Nacetylgalactosamine residues on ZP (area labeled by peanut agglutinin lectin, PNA) were assessed in IVM and in vivo matured (IVV) oocytes at different hours post insemination. After maturation, IVM and IVV oocytes displayed similar area occupied by cortical granules and it decreased in fertilized oocytes compared to unfertilized ones. Cortical reaction was influenced by boar and was faster in polyspermic than in monospermic oocytes, and in IVM than in IVV oocytes. The outer ZP of inseminated oocytes appeared stained by PNA and the labeled area increased along with gamete coculture time. This labeling was also observed after insemination of isolated ZP, indicating that this modification in ZP carbohydrates is not induced by cortical reaction. The steady and maintained cortical reaction observed at 4 to 5 h post insemination in IVV monospermic oocytes might reflect the physiological time course of this important event in pigs. Both maturation conditions and boar affect cortical granules release.

Following gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. The specific cortical granule-derived proteins responsible for these post-fertilization events are not fully characterized. Calreticulin, a highly conserved ubiquitous protein of 60 kDa, was exocytosed from activated hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Preliminary results from our laboratory have shown that calreticulin is located in the cortical area of pig oocytes (data not shown). This study was designed to test whether calreticulin is exocytosed after oocyte activation with calcium ionophore. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with 0.5% pronase in Ca2+-free PBS. After washing, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM) for 2min, transferred to a 100-μL droplet of exudate medium (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst) and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. After incubation, the medium containing the oocyte exudate (n = 1000) was carefully aspirated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The gel was then electro transferred onto a polyvinylidene fluoride (PVDF) membrane, incubated with an anti-calreticulin rabbit polyclonal antibody (1:1000) and finally conjugated to horseradish peroxidase (1:20 000) for 1 h with a monoclonal anti-rabbit IgG. Membrane visualization was accomplished using the ECL plus method and Typhoon 9410. A control group was performed with exudate collected from non-activated ZP-free oocytes. To verify cortical reaction and calreticulin exocytosis, an aliquot of activated ZP-free oocytes (n = 18) were fixed (3.7% paraformaldehyde for 30 min), permeabilized (0.1% Triton X-100 for 10 min), incubated with anti-calreticulin antibody (1:10 for 1 h) and conjugated to tetramethyl rhodamine isothiocyanate (1:400 for 1 h) with an anti-rabbit IgG. Finally, samples were incubated with peanut agglutinin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min), mounted and examined under a confocal microscope. No statistical analysis was made because the observations were purely qualitative. A Western blot analysis showed an immunoreactive band of ∼60 kDa, consistent with the expected size of calreticulin, in the lane containing the exudate from activated oocytes. No band was observed in the lane with the exudate collected from non-activated oocytes. Observation under confocal microscopy showed no PNA or anti-calreticulin fluorescence in the cortical region, indicating that the activated pig oocytes displayed full cortical reaction and calreticulin exocytosis during incubation time. These results show that calreticulin protein is exocytosed after the chemical activation of ZP-free pig oocytes as well as the disappearance of the cortical granule monolayer. The possible role of calreticulin on preventing polyspermy should be further investigated. Supported by MEC and FEDER (AGL2009-12512-C02-01) and CONACYT (0105961/I0110/194/09).

International audience; The aim of this study was to investigate the process of the cortical reaction in eggs of pikeperch, Sander lucioperca (L.), as well as the application of microscopic assessment of this process in egg quality evaluation. The analysis was carried out with eggs obtained from 10 females by artificial reproduction, in which hormonal stimulation with hCG was applied. Subsequently, each sample of eggs (separately from each female fish) was analysed. The analysis included observation of the cortical reaction and the process of egg swelling, and determination of the effect of temperature (12, 14 and 16 degrees C) and the presence of spermatozoa on the cortical reaction. The results indicate that the cortical reaction in pikeperch eggs is quite violent, resulting in visible deformation of eggs between 3 and 5 min after activation. No effect of temperature or the presence of spermatozoa on the cortical reaction was observed. A strong correlation was recorded for the percentage of egg deformations observed and embryo survival rate. The described method of determination of pikeperch egg quality (based on egg deformation rate between 3 and 5 min after activation) may be highly useful, both in scientific research (where high-quality eggs are required) and in hatchery practice.

The cortical reaction is a mechanism that prevents polyspermy by cortical granule content being released into the periviteline space, modifying the zona pellucida (ZP). Knowledge about specific cortical granule-derived proteins has progressed slowly because these organelles contain only picogram quantities of proteins. An efficient method for collecting cortical granule content would help in its study; chemical activation of ZP-free oocytes has been successfully used in the murine model (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Calcium ionophore A23187 is an effective chemical stimulator for provoking the cortical reaction in ZP-intact pig oocytes. However, the commonly used protocol (50 μM for 5min) cannot be employed with ZP-free oocytes because the oolemma is damaged, oocyte lysed and medium contaminated with ooplasm content, which is necessary to reduce the time and ionophore concentration (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst). The objective of this study was to evaluate the efficiency of this activation protocol for provoking the cortical reaction in ZP-free oocytes by assessment with confocal and electron microscopy. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with pronase. Then, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM for 2min), transferred to an exudate medium and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. Control ZP-free oocytes were incubated without being activated. After incubation, ionophore-treated (n = 10) and control oocytes (n = 18) were used to assess the presence of a cortical granule monolayer. An aliquot was fixed, permeabilized (0.1% Triton), incubated with peanut agglutinin lectin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min) and examined under a confocal microscope. Presence or absence of a cortical granule monolayer at the equator level was recorded. Another aliquot was fixed and processed for electron microscopy observation. The cortical granules in the whole oocytes were counted and results are presented as the mean ± standard error of the mean. No cell lysis was observed in control or activated ZP-free oocytes after treatment and incubation time. The confocal study showed that the activation protocol provokes a full cortical reaction in 100% of A23187-treated oocytes, given that no peanut agglutinin labeling was observed in the cortical area. Presence of a cortical granule monolayer under the oolemma was observed in 100% of control oocytes. Cortical granule release was confirmed by electron microscopy. Control oocytes had 5.90 ± 1.78 cortical granules per 5 μm of oolemma, whereas activated oocytes exhibited a significant reduction (P

No cleavage (nc) is a maternal effect mutant gene, recessive and sex limited. It affects the eggs laid by homozygous mutant females, independently of the male genotype. Contrary to normal oocytes, following germinal vesicle breakdown (GVBD) during maturation, the transient microtubular array (TMA) is not formed, nor are the meiotic spindles. Cytoplasmic asters with condensed chromosomes are present in the majority of oocytes, as well as microtubular bundles and sometimes cytoplasmic spindle-like asters. These mature oocytes exhibit a disturbance in yolk platelet arrangements. The white spot is rather irregular, and the maturation period is longer than normal. Transfers of cytoplasm from nc mature oocytes into normal stage VI oocytes resulted in abnormal maturation of the normal oocytes. Reciprocal transfers (cytoplasm from normal mature oocytes into nc stage VI oocytes) induce the formation of spindles, usually cytoplasmic; this indicates that the deficiency can be partly rescued. Following fertilization, the nc eggs show neither contraction nor rotation; polyspermy is present in the majority of cases. Even in the same egg, simultaneous spindles and nuclei can be observed, revealing a disturbance in the spatial localization of regulators of the cell cycle. Cytokinesis never occurs. Polyspermy results from the absence of cortical reaction following sperm entry. However, when mature nc oocytes are treated with PMA, they show cortical granule exocytosis and the formation of an altered vitelline envelope. The different factors possibly involved in these anomalies are discussed in relation to cytoarchitectural disorganization of the cell and abnormal cell cycle regulation. © 1994 Wiley-Liss, Inc.

To determine the temporal relationship between cortical granule exocytosis and the repetitive calcium transients, which are characteristic of mammalian fertilization, we monitored membrane addition from exocytosis during fertilization of hamster eggs. Continuous measurement of membrane capacitance by applying a 3.1-nA alternating current at 375 Hz showed addition of cortical granule membrane. Simultaneous measurement of membrane potential revealed each calcium transient by the appearance of transient hyperpolarizing responses due to calcium-activated potassium channels in the egg. The initial membrane capacitance of the eggs averaged 736 +/- 44 pF (mean +/- SD; n = 7) and an increase in capacitance of 61 +/- 19 pF occurred within 4 sec of the start of the first hyperpolarizing response (HR) after fertilization. Immediately after the first increase in capacitance there was a gradual decline in membrane capacitance in all eggs and in five/seven eggs the capacitance returned to the unfertilized level in 7.8 +/- 4.4 min. The gradual decline in capacitance after the first increase indicated endocytosis, which was confirmed by the internalization of fluorescently labeled dextran. Superimposed on the gradual decline in membrane capacitance were smaller increases in capacitance that occurred with the second and later HRs. The total increase in capacitance from the first three events averaged 72 +/- 19 pF, representing an average increase in capacitance of about 10% of the capacitance of the unfertilized egg. By labeling eggs before and after permeabilization with two different fluorochromes attached to Lens culinaris agglutinin, we demonstrate that the dispersal of the cortical granules contents does not occur immediately after exocytosis. Our results demonstrate that cortical granule exocytosis in hamster eggs is closely coupled to the periodic increases in calcium, that the contents of the cortical granules are slow to disperse, and that after exocytosis, the surface area of the egg returns to the unfertilized level because of a period of endocytosis.

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs. Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).

Cortical granules are membrane bound organelles located in the cortex of unfertilized oocytes. Following fertilization, cortical granules undergo exocytosis to release their contents into the perivitelline space. This secretory process, which is calcium dependent and SNARE protein-mediated pathway, is known as the cortical reaction. After exocytosis, the released cortical granule proteins are responsible for blocking polyspermy by modifying the oocytes' extracellular matrices, such as the zona pellucida in mammals. Mammalian cortical granules range in size from 0.2 um to 0.6 um in diameter and different from most other regulatory secretory organelles in that they are not renewed once released. These granules are only synthesized in female germ cells and transform an egg upon sperm entry; therefore, this unique cellular structure has inherent interest for our understanding of the biology of fertilization. Cortical granules are long thought to be static and awaiting in the cortex of unfertilized oocytes to be stimulated undergoing exocytosis upon gamete fusion. Not till recently, the dynamic nature of cortical granules is appreciated and understood. The latest studies of mammalian cortical granules document that this organelle is not only biochemically heterogeneous, but also displays complex distribution during oocyte development. Interestingly, some cortical granules undergo exocytosis prior to fertilization; and a number of granule components function beyond the time of fertilization in regulating embryonic cleavage and preimplantation development, demonstrating their functional significance in fertilization as well as early embryonic development. The following review will present studies that investigate the biology of cortical granules and will also discuss new findings that uncover the dynamic aspect of this organelle in mammals.

The involvement of calcium- or protein kinase C (PKC)-dependent pathways in cortical granule exocytosis (CGE) and pronucleus formation was examined in mouse eggs using the specific PKC stimulator OAG (1-oleyl-2-acetyl-sn-glycerol) at different external calcium concentrations ([Ca2+]e) ranging from 1.7mM to 0.1μM. A 10 min exposure of eggs to 150 μM OAG in the presence of 1.7mM [Ca2+ecaused a large calcium influx, cortical granule release and 82% activation. The increased permeability of the egg membrane to Ca2+ions after OAG treatment lasted 20 min. At [Ca2+]elower than 1.7 mM, both OAG-induced calcium influx and CGE decreased, reaching a non-detectable level at 0.1 μM and 100 μM [Ca2+]e, respectively. Resumption of meiosis was not affected by [Ca2+]eabove 200 μM but it was reduced at any lower [Ca2+]e, with a minimum activation frequency of 46% at 0.1 μM [Ca2+]e. Loading of eggs with ≥3μM of the calcium chelator BAPTA AM (1,2-bis(o-aminophenox- y)ethane-N′,N′,N′,N′tetraacetic acid-acetoxymethyl ester) prior to OAG treatment caused a reduction in meiosis resumption with 50% of eggs forming pronuclei. Potent inhibitors of PKC, such as acridine orange and sphingosine, did not interfere with OAG-induced CGE. Conversely, these compounds prevented OAG-induced pronucleus formation in a dose-dependent manner with an IC50(inhibiting concentration, 50%) of 5μM and 30 μM for acridine orange and sphingosine, respectively. Microinjection of inositol 1,4,5-trisphosphate into eggs at 0.1 pM elicited Ca2+release from intracellular stores and the cortical reaction, but failed to stimulate pronucleus formation. These results indicate that, in mouse eggs, CGE is a PKC-independent event, and that the transition from M-phase to interphase may require PKC activity for stimulation.

In order to achieve fertilization sperm cells first need to successfully interact with the zona pellucida. Before reaching the zona pellucida the sperm cell undergoes extensive remodeling both in the male and female genital tract. These changes serve to mediate optimal recognition of the zona pellucida in the oviduct (primary zona pellucida binding). Optimal sperm-zona interactions are crucial for porcine oocyte fertilization: The zona pellucida- attached sperm cell is triggered to undergo the acrosome reaction and will also become hypermotile. Together these two responses allow the sperm cell to drill through the zona pellucida (secondary zona pellucida binding) this coincides with local sperm zona drilling so that a few sperm can reach the perivitellin space. This delaying strategy allows only one sperm cell in a given time-point to bind and fuse with the oocyte (fertilization) and thus minimizes the risk of polyspermy. The polyspermy block is essentially executed by the fertilized oocyte that immediately secretes its cortical granule content into the perivitellin space. This content blocks sperm-oocyte fusion either by sticking to the oolemma or by the induction of a biochemical reaction of the zona pellucida (zona pellucida hardening). The cortical reaction thus blocks sperm-zona pellucida binding and/or sperm-zona pellucida drilling. Note that zona pellcuida interactions under pig IVF conditions relatively frequently result in polyspermy. It is not clear whether this is also the case in vivo after natural mating. More importantly we do not know how other artificial reproductive technologies affect polyspermy rates. This is especially relevant for new sperm treatments and insemination technologies in which sperm are activated by capacitation media essentially mimicking the IVF media. Therefore, better understanding of sperm activation and of the arrangement of proteins involved in zona pellucida interactions are relevant for designing strategies to further improve pig reproduction.

The sequence and timing of morphological changes during envelope formation was followed in diapause eggs of Pontella mediterranea (Crustacea, Copepoda). The multilayer coat enveloping these eggs resulted from the exocytosis of 4 types of cortical vesicles that sequentially released their contents in the perivitelline space. These included small high-density vesicles (hDV) with electron-dense material, vesicles (V) with dense ring granules and a uniform matrix contained within the same compartment, large high-density (HDV) vesicles, and large moderately dense (MDV) vesicles. All of these cortical vesicles were present in newly spawned, fertilized eggs. Their exocytosis resulted from egg activation. One of these cortical vesicles (V) was similar in morphology to the intracisternal granules precursors of endogenous yolk. Intracisternal granules, characteristic of previtellogenic oocytes of many crustaceans, were present in previtellogenic oocytes of P. mediterranea but disappeared in later stages of oocyte development once yolk formation was completed. We discuss the role of cortical vesicles in the formation of the complex extracellar coat enveloping copepod diapause eggs. © 1992 Wiley-Liss, Inc.

Pig oocytes were matured in vitro in a modified M-199 medium for 44 h, subjected to electrical stimulation and scored for activation 6 h later. Sham pulsed oocytes, exposed to electroporation medium and an a.c. field, did not develop the female pronucleus any more frequently than occurs spontaneously (8.3% within 50 h of culture). However, a single d.c. pulse proved extremely efficient in activating pig oocytes. Pulses of 0.75-1.65 kV cm-1 lasting 30 or 100 microseconds activated at least 90% of matured oocytes. The developmental pathway taken by the activated oocytes depended on the parameters of the pulse. The lowest effective stimulation (0.45 and 0.60 kV cm-1 for 30 microseconds) frequently produced oocytes that remained in pre-pronuclear stages of activation (29.4 and 42.3%, respectively). Extrusion of the second polar body and creation of one pronucleus was the most frequent type of activation (in up to 88.2% among the activated oocytes). The strongest stimulations used (1.05-1.65 kV cm-1 for 100 microseconds) often yielded oocytes that failed to extrude the second polar body and formed two or more pronuclei (up to 56.3%). Under optimal stimulation (0.75 kV cm-1), the activated oocytes proceed synchronously to interphase of the first mitotic division. Anaphase II is reached within 30 min and telophase Ii at 1 h after application of the pulse. The second polar body is extruded about 2 h after activation. Well-defined swelling pronuclei were found in oocytes 5-6 h after activation. The relationship between the stage of oocyte maturation and susceptibility to activation was investigated. The period of culture in which the oocytes develop the activation competence (32-36 h of culture) overlapped with the period in which the oocytes complete meiosis (28-38 h). This suggests that ageing in meiotic arrest is not essential for pig oocytes to become activated by electric pulses. Activation of pig oocytes was accompanied by release of cortical granules. In sections of control (metaphase II) oocytes, an average of 7.3 intact cortical granules per 10 microns of overlying cytoplasmic membrane was found. This number dropped to 1.5 in 10 microns within 30 min after the pulse.

The experiments compare intracellular changes in porcine eggs induced by electrical activation with those induced by sperm penetration. Adequate electrostimulation induces changes in both cortical granule exocytosis and protein synthesis similar to those induced by sperm during fertilization. However, ionic changes induced by electrostimulation differ markedly from those initiated at fertilization. Thus, dynamic video imaging using Fura-2 as a Ca2+ probe provides evidence that parthenogenetic activation induced by electrostimulation is initiated by a single sharp rise in the concentration of intracellular free calcium ([Ca2+]i) in the egg. The intracellular Ca2+ transient increase is triggered by an influx of extracellular Ca2+ immediately after elec-trostimulation. The amplitude of the intracellular Ca2+ transient increase is a function both of the extracellular Ca2+ concentration and of electric field parameters (field strength and pulse duration). Imaging demonstrates further that a single electrical pulse can only induce a single Ca2+ transient which usually lasts three to five minutes; no further Ca2+ transients are observed unless additional electrical stimuli are applied. By contrast, sperm-induced activation is characterised by a series of Ca2+ spikes which continue for at least 3 hours after sperm-egg fusion. The pattern of Ca2+ spiking after fertilization is not consistent during this period but changes both in frequency and amplitude. Overall, the results demonstrate that, although electrostimulation induces both cortical granule exocytosis and protein reprogramming in porcine eggs, it does not reproduce the pattern of [Ca2+]i changes induced by sperm entry at fertilization.

Publisher Summary This chapter discusses the biochemical nature of the elastic element in the cortex. The discovery of two distinct assembly states of actin in the prefertilization cortex and the purification of numerous actin-associated proteins that are likely to regulate these cortical changes provide appropriate materials for future studies. The applications of cryofixation techniques and lowicryl embedding methods now allow the localization of the various actin-associated proteins as a function of time after fertilization. Such structural studies provide important clues regarding the possible involvement of these proteins in the fertilization-induced changes in actin organization in the sea urchin egg cortex.

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.

Surface changes induced by sea water were analyzed in the ovulated oocyte of the prawn Palaemon serratus. They depended on the presence of external Mg2+ but not on external Ca2+ alone. Increasing external Mg2+ from 0 mM to 30 mM stimulated first a progressive disappearance of preexisting microvilli, which was over within 30 min of incubation. This is correlated with membrane removal via internalization of coated vesicles, ascertained by observations of endocytosis of an extracellular fluid-phase marker and by measurement of a diminution in membrane capacitance (Cm). Thirty-five minutes after sea water contact, the prawn oocyte underwent a cortical reaction independent of fertilization. It consists in a heavy exocytosis of ring-shaped elements, leading to the deposition of a thick capsule, and requiring a threshold Mg2+ concentration of greater than or equal to 10 mM and at least a 3-min incubation with Mg2+. Concurrently, the values of the membrane capacitance (Cm) and conductance (Gm) increased about 2 and 10 times their initial values, respectively. The calcium ionophore ionomycin, added to Mg(2+)-free artificial sea water, stimulated the cortical reaction with requirement of external Ca2+. Other divalent cations (Mn2+, Zn2+, Co2+, Ni2+, Cd2+) instead of Mg2+, induced the cortical reaction, but Ba2+, Sr2+, and La3+ did not. When eggs are fertilized, the cortical reaction takes place in two steps, the first being a discrete exocytosis of a foamy material and the second always involving ring-shaped elements.

We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/micron2 and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form dome-shaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).

Although activation of a sea urchin egg by sperm leads to three phases of membrane conductance increase in the egg, the mechanism by which the sperm causes these conductance changes is not known. We used the loose patch clamp technique to localize the conductance changes in voltage clamped eggs. A patch of the egg's membrane was isolated from the bath by pressing the loose patch clamp pipette against the egg surface. Sperm added to the bath attached to the surface of the egg in a region other than at the isolated membrane patch. During phase 1 of the activation current, no changes of the membrane conductance were detected. At the time of, and subsequent to the onset of phase 2, large currents recorded between the interior of the patch pipette and the bath were attributed to changes of the seal resistance between the surface of the egg and the pipette. A local change of membrane conductance was observed during phase 2 despite the changes of seal resistance. During phase 2, the large amplitude and short duration of the local membrane conductance increase relative to the membrane, conductance increase for the whole egg during phase 2 indicated that the conductance increase occurred over the entire surface of the egg, but not simultaneously. The time when the peak conductance for the membrane patch occurred, relative to the time of onset for phase 2 in the whole egg, depended on the distance, measured in a straight line, between the site of sperm attachment and the tip of the pipette. These data indicate that the localized conductance increase progressed over the surface of the egg from the site of sperm attachment to the opposite pole of the egg. It is proposed that the local conductance increase, the cortical reaction, and the change of seal resistance are all evoked by a common cytoplasmic message that progresses throughout the cytoplasm of the egg from the site of sperm attachment to the opposite pole of the egg.

We report that parthenogenetic activation (pronuclear formation) is induced during in vitro culture of recently ovulated (13-14 hr post-hCG) mouse oocytes in pyruvate deficient medium. Pronuclear formation occurred when oocytes were cultured in medium containing 1/10X (Pyr-) or lower concentrations of pyruvate but failed to occur either in oocytes cultured in the presence of 0.47 mM (1X, Pyr+) or 1/2X pyruvate or in oocytes cultured in the absence of pyruvate but with cumulus cells. Pronuclear formation was evident within 8 hr of culture and completed by 16 hr and remained intact during continuous culture in Pyr- medium. Transfer of pronuclear oocytes to Pyr+ medium resulted in pronuclear membrane disassembly and further parthenogenetic development. A similar incidence of parthenogenetic activation occurred when recently ovulated oocytes were cultured in the presence of cycloheximide but not following ethanol or hyaluronidase treatment. However, both ethanol and hyaluronidase induced pronuclear formation in in vivo aged oocytes. Results suggest that the type of activation induced varies with the age of the oocyte and the nature of the stimulus. Amino acid uptake ([35S]methionine) by oocytes was unaffected by Pyr- culture whereas incorporation into protein was markedly inhibited. Gel electrophoretic analysis of labeled egg extracts revealed a marked inhibition of egg protein synthesis after 4 hr of culture in Pyr-. The occurrence of a cortical reaction was monitored by binding of fluorescent labeled lectin to the oocyte surface. A cortical reaction occurred in response to ethanol treatment of freshly ovulated and in vivo aged oocytes cultured in Pyr+ medium but not in pronucleate oocytes induced by Pyr- culture. Suppression of ethanol-induced cortical reaction by Pyr- culture was restored following transfer of oocytes to Pyr+ medium. Results demonstrate that nuclear events as well as plasma membrane events can be simply regulated by controlling the amount of energy substrate available to the germ cell. Effects of Pyr- culture in inducing pronuclear formation appear to be mediated in a large part via inhibition of protein synthesis.

Publisher Summary The extracellular matrix (ECM) surrounding amphibian eggs is composed of jelly coat layers, the egg envelope, and the fibrous elements of the perivitelline space. The oviposited egg envelope provides an initially penetrable structure to the sperm which is subsequently modified by products released in the cortical reaction to a sperm-impenetrable structure in the block to polyspermy reaction. At fertilization the egg envelope is chemically modified and its macromolecular permeability properties changed. This permeability change (hardening) results in an osmotically driven envelope elevation, owing to the influx of water into the perivitelline space. The jelly after fertilization functions as a “sticky substrate” for the adherence of the zygote to objects in its surroundings, protects the zygote against physical damage, and also provides a microbiological barrier (bacteria are rarely found within the jelly coat layers). Thus, after fertilization the ECM protects the developing embryo and functions as a barrier for the chemical and biological regulation of the embryo environment. This protective function of the ECM persists until the embryo develops into a free swimming tadpole and hatches from the ECM. Thus, the chapter briefly discusses isolation of ECM structures from Xenopus laevis oocytes, eggs, and embryos.

Cortical granules (CG) are clue organelles in the oocyte since their content is released under oocyte activation (i.e. fertilization) modifying the zona pellucida and thus blocking polyspermy. Once released, CG are not renewed. Research on cortical reaction and putative CG enzymes has progressed slowly because mammalian eggs contain only picogram quantities of CG-derived proteins (Moller and Wassarman 1989 Dev. Biol. 132, 103–112; Green 1997 Rev. Reprod. 2, 147–156), so the protein(s) responsible for the physiological changes in ZP after cortical reaction are not well known. The objective of this project was to study porcine CG content in in vitro-matured porcine oocytes by means of lectin precipitation with peanut agglutinin (PNA), since this lectin binds to porcine CG (Yoshida et al. 1993 Mol. Reprod. Dev. 36, 462–468). Immature porcine cumulus–oocytes complexes (COCs) from Landrace � Large White gilts were in vitro-matured for 44 h in NCSU-37 medium. After IVM period, COCs were stripped of cumulus cells, washed in PBS, and quickly washed through purified water. Then oocytes were lysed in a fresh water droplet by gentle pipetting using a narrow-bore glass pipette. Once lysed, zonae pellucidae were removed and oocyte cytoplasmic content (lysate) collected. Lysate from 1000 IVM-oocytes was incubated under continuous shaking (2 h, room temperature) with 100 µL PNA-agarose (Sigma, St. Louis, MO, USA) so that proteins bound to PNA could be precipitated by centrifugation. After lectin precipitation, proteins were detached from PNA-agarose beads by boiling in reducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (5 min, 100�C) (Laemmli 1970 Nature 227, 689). Samples were then centrifuged (5 min, 7000g), the pellet containing PNA-agarose beads was discarded, and the supernatant containing the proteins was collected and further separated by SDS-PAGE. The silver staining of electrophoresis gels revealed eleven bands from 37 to 180 kDa, so a second gel was electrotransferred to a polyvinylidene fluoride (PVDF) membrane (100V, 1 h) and incubated with PNA-horseradish peroxidase (PNA-HRP, 10 µg mL–1) for 1 h. Visualization was accomplished using the enhanced chemiluminiscence (ECL plus) method and Typhoon 9410 following the manufacturer's instructions (Amersham Biosciences, Freiburg, Germany); only four bands of approximately 57 kDa, 60 kDa, 70 kDa, and 80 kDa were observed. These bands will be cut and processed for proteomic analysis for further studies. Preliminary results show that porcine CG-derived proteins can be studied by PNA lectin precipitation. These results could be employed in the future to develop specific antibodies against porcine CG.

At fertilization the sea urchin egg undergoes a dramatic structural reorganization of its surface. Within the first few seconds there is a depolarization of the egg plasma membrane and this is followed by a transient increase in intracellular Ca++. The elevated Ca induces the exocytosis of cortical granules and the resulting fertilization membrane helps make the egg refractory to further insemination.

Alberto Monroy, together with Kao, Grundfest and Tyler, were amongst the first to recognize the importance of ion fluxes through the plasma membrane in the process of egg activation (Tyler et. al. 1956). In the 1970’s the electrical events at activation in sea urchin eggs were recorded (Steinhardt et al 1971; Ito and Yoshioka, 1973; Jaffe, 1976 and Chambers and de Armendi, 1979), and shortly afterwards measurements were extended to fish, amphibia and mammals (see Igusa et al., 1983; McCulloh et al., 1983; Nuccitelli, 1980; Talevi et al., 1985 for references). The sea urchin response was noted to be a biphasic depolarization (Ito and Yoshioka, 1973). However, neither the biophysical origin of this depolarization, nor the mechanism by which it was triggered, were known. Here we follow the progress made in this field and describe how electrical measurements have been utilized to elucidate the mechanism of egg activation.

The fertilization pathway in mice consists of several steps that must occur in a compulsory order prior to fusion of sperm and egg (reviewed in Wassarman, 1987a,b, 1988a). The steps include: (i) loose “attachment” and then tight species-specific “binding” of acrosomeintact sperm to the unfertilized egg’s extracellular coat, or zona pellucida; (ii) completion of the acrosome reaction, an exocytotic event involving fusion of outer acrosomal and plasma membranes, by bound sperm; (iii) penetration of the zona pellucida by bound acrosomereacted sperm (perhaps, by using a sperm proteinase, “acrosin”); (iv) fusion of acrosomereacted sperm with the egg’s plasma membrane. Within a few minutes of sperm-egg fusion, the zona pellucida is altered (“zona reaction”) such that it becomes refractory to both binding of sperm and penetration by sperm; this constitutes a secondary block to polyspermy. These changes in the nature of the zona pellucida are thought to be brought about by cortical granule enzymes released into the extracellular coat as a consequence of the cortical reaction (fusion of cortical granule and plasma membranes). Thus, the steps in the fertilization pathway include species-specific cellular recognition, intracellular and intercellular membrane fusions, and enzyme-catalyzed modifications of cellular investments.

The newly laid egg of Carcinus maenas is attached to a maternal ovigerous seta by a funiculus which consists of the two superimposed vitelline envelopes 1a + 1b, highly stretched and concurrently showing important structural alterations. The funiculus is glued to the specialized seta merely owing to the strong adhesiveness of its external face comprising the outermost vitelline envelope 1a, without any added adhesive. The subjacent envelope 2, originated from the cortical reaction, is not involved in such a funiculus elaboration. In the course of the embryonic development, four new coatings are successively secreted from the ectodermal embryonic cells, underneath the (1a + 1b + 2) fertilization envelope or embryonic capsule. They will remain until hatching in this concentric order, thus giving evidence of successive embryonic moulting cycles, with apolysis but without exuviation. In addition, the successive secretory phases, regarding to the embryonic envelope elaborations, happen in presence of high concentrations of the ecdysteroid ponasterone A which might be involved consequently in such secretory processes.

We have examined the formation of the egg attachment stalk in the lobsters Homarus americanus and H. gammarus. The formation of the stalk is similar in both species. Ovulated oocytes are surrounded by a single coat, envelope 1, composed of layers 1A and 1B. After passage through the gonopore and exposure to sea water, envelope 1 swells and becomes sticky. A second coat, envelope 2, forms between the oocyte and envelope 1 during a complex cortical reaction initiated after fertilization. Eggs pass over the ventral surface of spawning females to the region of the pleopods, where they stick by means of layer 1A to each other and to the ovigerous setae. Layers 1A and 1B are soft and pliable at this time. During egg attachment, the pleopods beat vigorously and cause envelope 1 to stretch and form attachment stalks. Beating probably also causes the attachment stalks to twist and wrap around the ovigerous setae. After the egg mass is secured to the ovigerous setae, envelope 1 of both the attachment stalk and egg coat condenses to form a tough material capable of securing the egg mass to the pleopods for intervals up to 16 months. After larvae hatch, portions of the egg coats and the attachment stalks are retained on the ovigerous setae until the female undergoes her next molt.

During the cortical reaction, Carcinus maenas eggs successively released a fine granular material and a massive amount of ring-shaped elements that subsequently formed most of the fertilization envelope. The ring-shaped elements came from egg cortical vesicles and, owing to their striking morphology, acted as naturally occurring markers in ultrathin sections, which permitted us to understand the pathway of their intracellular transport. In this respect it was established that the ring-shaped elements and their enclosing vesicles originated in the endoplasmic reticulum both in ripe oocytes and early fertilized eggs maintained under in vitro conditions. The intracellular transport pathway of the endoplasmic reticulum-derived vesicles seemed to bypass the Golgi apparatus. Accordingly, the ring-shaped elements appeared to be released by direct exocytosis from the endoplasmic reticulum system. Finally, a tentative scheme of oocytes functioning is suggested for crustacean decapods, based on the remarkable similarities between the structure and ER origin of the ring-shaped elements involved in the cortical reaction and the disc-shaped granules traditionally considered as endogenous yolk precursors. The scheme implies that the oocyte ER system might produce a precursor common to the cortical reaction exudate and to the endogenous yolk, in the form of the ring- or disc-shaped elements.

We have analysed in detail the Na + content and Na + influx during fertilization and first divisions of the sea urchin egg ( Paracentrotus lividus ) using a filtration technique devised to eliminate rapidly contamination by the Na + of external sea water. In the first 5 min following fertilization the egg fills up with Na + (+ 30%). Thereafter Na + is extruded and the Na + content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na + content is due to a large increase in Na + influx already detected at 20 sec. The Na + influx reaches its maximum at 1 min and its minimum at 5 min. H + excretion follows the same kinetics. A second increase in Na + influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na + influx were observed for the first three divisions. Fertilization also immediately stimulates the Na + efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na + content of the embryos. Activation of the eggs by weak amine bases (5 mM NH 4 Cl) which bypasses the early cortical reaction produces only a depletion in the Na + content of the egg similar to that produced by fertilization. NH 4 Cl also increases the Na + influx soon after fertilization, although no transient variations are noted.

The drugs, fluphenazine, chlorpromazine, dibucaine, propranolol, vinblastine and W7[N-(6-arninohexyl)-5 chloro-1-napthalene-sulfonamide], which have been shown to prevent formation of the ternary activated complex of Ca++-calmodulin with several soluble or membrane proteins, inhibit the cortical reaction induced by fertilization, by ionophore A 23187 or by the microinjection of Ca++ buffers when applied from outside to sea urchin eggs. In contrast, direct intracellular microinjection of these drugs, even at concentrations much exceeding their I50 for external application, does not suppress elevation of the fertilization membrane, although it prevents cleavage after fertilization. The implication is that intracellular calmodulin is not the receptor of Ca++ in the Ca++-dependent exocytosis of cortical granules induced by fertilization, by ionophore, or by the micro-injection of calcium buffers.

Activation of the teleost (Brachydanio) fish egg includes the exocytosis of cortical granules, the construction of a mosaic surface consisting of the unfertilized egg plasma membrane and the limiting membranes of the cortical granules, and the appearance of coated and smooth vesicles in the cytoplasm (Donovan and Hart, '82). Unfertilized and activated eggs were incubated in selected extracellular tracers to (1) determine experimentally if cortical granule exocytosis was coupled with the endocytosis of membrane during the cortical reaction, and (2) establish the intracellular pathway(s) by which internalized vesicles were processed. Unfertilized eggs incubated in dechlorinated tap water or Fish Ringer's solution containing either horseradish peroxidase (HRP; 10 mg/ml), native ferritin (12.5 mg/ml), or cationized ferritin (12.5 mg/ml) were activated as judged by cortical granule breakdown and elevation of the chorion. Cells treated with HRP and native ferritin exhibited a delay in cortical granule exocytosis when compared with water-activated eggs lacking the tracer. Each tracer was internalized through the formation of a coated vesicle from a coated pit. Since coated pits appeared to be topographically restricted to the perigranular membrane domain of the mosaic egg surface, their labeling, particularly with cationized ferritin, strongly suggested that the retrieved membrane was of cortical granule origin. Cationized ferritin and concanavalin A (Con A) coupled with either hemocyanin or ferritin labeled the surface of the unactivated egg and both domains of the mosaic egg surface. Transformation of the deep evacuated cortical granule crypt into later profiles of exocytosis was accompanied by increased Con A binding. Within activated egg cortices, HRP reaction product, native ferritin, and cationized ferritin were routinely localized in smooth vesicles, multivesicular bodies, and autophagic vacuoles. Occasionally, each tracer was found in small coated vesicles adjacent to the Golgi and within Golgi cisternae. The intracellular distribution of HRP, native ferritin, and cationized ferritin suggests that internalized membrane is primarily processed by organelles of the lysosomal compartment. A second and less significant pathway is the Golgi complex.