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Context: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. Aim: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus–oocyte complexes (COC) to improve oocyte metabolism. Methods: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Key results: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. Conclusions: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. Implications: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs. In vitro -produced embryos are essential for employing assisted reproductive technologies to establish porcine models. However, polyspermy remains a major obstacle to the production of porcine embryos in vitro. This study was conducted to test a coculture system of porcine luteal cells during in vitro maturation to improve suboptimal conditions. Our model could be an alternative to replace the conventional maturation medium, affecting the metabolism of the oocytes and leading to lower rates of polyspermy and higher rates of embryo development. Image by Gabriela Teplitz and Alexandre Bastien. [ABSTRACT FROM AUTHOR]

Purpose. To analyze the scientific data of domestic and foreign authors regarding the peculiarities of artificial reproduction of sturgeonss (Acipenseriformes). To investigate the state of study of the phenomenon of polyspermic fertilization in sturgeons and the factors that induce it in the conditions of artificial reproduction and cultivation. Findings. A review analysis of the results of scientific studies by domestic and foreign authors was carried out on the pecularities of artificial reproduction of sturgeons and the phenomenon of polysperm fertilization of their eggs in controlled conditions of aquaculture. It is shown that information on the problems of sturgeon polyspermy and its induction by various factors was practically absent in the available professional literature in recent years. Since during the artificial reproduction of sturgeons, a significant number of embryos have atypical development, which leads to their death even before hatching, it has been suggested that atypical forms of cell division were caused by polyspermy. The conducted studies added to the knowledge about the peculiarities of the structure of the sturgeon egg envelope and the processes that occur during fertilization. Despite the fact that there is a natural mechanism to prevent the possibility of polyspermic fertilization, this phenomenon occurs quite often, causing an impairment of zygote fragmentation at the initial stages of embryogenesis, and therefore a decrease in the yield of larvae. Understaning of the mechanism of blocking polyspermy in sturgeons and the factors that induce it requires further studies. The list of scientific articles includes 61 sources, 58 in English. The published publications mainly cover the period of the last twenty years. Practical value. A review of data from scientific and professional sources on the specified topic will allow a comprehensive approach to the understanding of the peculiarities of artificial reproduction of sturgeons, the phenomenon of polyspermy and increasing the yield of larvae in sturgeon farming. Review material on this topic can be interesting and useful for scientists, practitioners, students whose interests are related to aquaculture and used in the educational process of training specialists in specialty 207 “Aquatic bioresources and aquaculture”.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction.Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes.Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis.Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg's receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process. [ABSTRACT FROM AUTHOR]

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype–phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans.

In sea urchin, the immediate contact of the acrosome-reacted sperm with the egg surface triggers a series of structural and ionic changes in the egg cortex. Within one minute after sperm fuses with the egg plasma membrane, the cell membrane potential changes with the concurrent increases in intracellular Ca2+ levels. The consequent exocytosis of the cortical granules induces separation of the vitelline layer from the egg plasma membrane. While these cortical changes are presumed to prevent the fusion of additional sperm, the subsequent late phase (between 1 and 4 min after fertilization) is characterized by reorganization of the egg cortex and microvilli (elongation) and by the metabolic shift to activate de novo protein and DNA syntheses. The latter biosynthetic events are crucial for embryonic development. Previous studies suggested that the early phase of fertilization was not a prerequisite for these changes in the second phase since the increase in the intracellular pH induced by the exposure of unfertilized sea urchin eggs to ammonia seawater could start metabolic egg activation in the absence of the cortical granule exocytosis. In the present study, we have demonstrated that the incubation of unfertilized eggs in ammonia seawater induced considerable elongations of microvilli (containing actin filaments) as a consequence of the intracellular pH increase, which increased the egg’s receptivity to sperm and made the eggs polyspermic at fertilization despite the elevation of the fertilization envelope (FE). These eggs also displayed compromised Ca2+ signals at fertilization, as the amplitude of the cortical flash was significantly reduced and the elevated intracellular Ca2+ level declined much faster. These results have also highlighted the importance of the increased internal pH in regulating Ca2+ signaling and the microvillar actin cytoskeleton during the late phase of the fertilization process.

Summary: During sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy. [ABSTRACT FROM AUTHOR]

Oocyte in vitro maturation does not entirely support all the nuclear and cytoplasmic changes that occur physiologically, and it is poorly understood whether in vitro maturation affects the competence of cortical granules to secrete their content during cortical reaction. Here, we characterize cortical granule exocytosis (CGE) in live mouse oocytes activated by strontium chloride using the fluorescent lectin FITC-LCA. We compared the kinetic of CGE between ovulated (in vivo matured, IVO) and in vitro matured (IVM) mouse oocytes. Results show that: (1) IVM oocytes have a severely reduced response to strontium chloride; (2) the low response was confirmed by quantification of remnant cortical granules in permeabilized cells and by a novel method to quantify the exudate in non-permeabilized cells; (3) the kinetic of CGE in IVO oocytes was rapid and synchronous; (4) the kinetic of CGE in IVM oocytes was delayed and asynchronous; (5) cortical granules in IVM oocytes show an irregular limit in regards to the cortical granule free domain. We propose the analysis of CGE in live oocytes as a biological test to evaluate the competence of IVM mouse oocytes. This article has an associated First Person interview with the first author of the paper.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Klinsky, Wetten, Zanni-Ruiz, Pavarotti, Berberian and Michaut.)

Parthenotes are characterized by poor in vitro developmental potential either due to the ploidy status or the absence of paternal factors. In the present study, we demonstrate the beneficial role of spermderived factors (SDF) on the in vitro development of mouse parthenotes. Mature (MII) oocytes collected from superovulated Swiss albino mice were activated using strontium chloride (SrCl2) in the presence or absence of various concentrations of SDF in M16 medium. The presence of SDF in activation medium did not have any significant influence on the activation rate. However, a significant increase in the developmental potential of the embryos and increased blastocyst rate (P < 0.01) was observed at 50 µg/ml concentration. Furthermore, the activated oocytes from this group exhibited early cleavage and cortical distribution of cortical granules that was similar to that of normally fertilized zygotes. Culturing 2-cell stage parthenotes in the presence of SDF significantly improved the developmental potential (P < 0.05) indicating that they also play a significant role in embryo development. In conclusion, artificial activation of oocytes with SDF can improve the developmental potential of parthenotes in vitro. [ABSTRACT FROM AUTHOR]

Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. [ABSTRACT FROM AUTHOR]

Improper activation and swelling of in vitro produced eggs of European eel, Anguilla anguilla, has been shown to negatively affect embryonic development and hatching. We investigated this phenomenon by examining the effects of salinity and sea salt type on egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation, using natural seawater adjusted to different salinities, varied among female eels, but no consistent pattern emerged. Activation salinities between 30–40 practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters. Chorion diameters reached maximal values of 1642 ± 8 μm at 35 psu. A positive relationship was found between egg neutral buoyancy and activation salinity. Nine salt types were investigated as activation and incubation media. Five of these types induced a substantial perivitelline space (PVS), leading to large egg sizes, while the remaining four salt types resulted in smaller eggs. All salt types except NaCl treatments led to high fertilization rates and had no effect on fertilization success as well as egg neutral buoyancies at 7 h post-fertilization. The study points to the importance of considering ionic composition of the media when rearing fish eggs and further studies are encouraged. [ABSTRACT FROM PUBLISHER]

Recent findings have refined our thinking on sperm interactions with the cumulus-oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona-cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm-ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals. [ABSTRACT FROM AUTHOR]

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP. [ABSTRACT FROM AUTHOR]

There are 1 to 4 rows and five types of cortical alveoli in the cortex of the pallas ( Hemibarbus labeo) egg. From the outer to the inner of the cortex, the diameter of the cortical alveoli decreases gradually. We found a new structure named solicitation speckle at the low latitude of the animal pole and near the micropylar apparatus between the ovum envelope and cell membrane in the zygote of fish. The solicitation speckle similar to type I cortical alveoli was very purple in H.E. chromosome, and it had no obvious boundary with the ovum envelope and membrane. Moreover, no membrane around the solicitation speckle in TEM was found. In SEM, the solicitation speckle looked flocculent. During cortical reaction, the solicitation speckle played a very important function in arousing cortical reaction. Thirty-five seconds after fertilization, cortical alveoli began to break down near the low latitude of the animal pole. At the same time, the same thing happened near the micropylar apparatus before cortical reaction. Both starting points encountered and healed up at the vestibule of the micropylar apparatus. The cortical reaction that happened near the low latitude of the animal pole was another new pattern. The cortical reaction was divided into four parts that included latent period, developmental period, climactic period and declining period. In the latent period, no cortical alveoli were released. In the developmental period, a few cortical alveoli were released outside the cortex. In the climactic period, several cortical alveoli were inosculated into a big vesicle and released intensely. In the declining period, the type V cortical alveoli and the other remnant cortical alveoli were released. Five minutes after fertilization, cortical alveoli were released entirely in the animal pole. Five minutes after fertilization, all of the remnant cortical alveoli were released. This leads us to conclude that cortical reaction is induced by type I cortical alveoli, and the solicitation speckle is a volcanic chain reaction under water or the other lower osmotic pressure of fluids. The outer cortical reaction can accelerate the inner cortical reaction. While cortical alveoli releases in batches, the cell plasma membrane is reorganized over and over. No cortical alveoli were found below the micropylar tube where sperm enters the ovum, which suggests that the cortical reaction prevents polyspermy. [ABSTRACT FROM AUTHOR]

Fertilization in mammals is initiated by species-restricted binding of free-swimming sperm to the unfertilized egg's thick extracellular matrix, the zona pellucida (ZP). Both acrosome-intact and acrosome-reacted sperm can bind to the ZP, but only the latter can penetrate the ZP, reach the egg's plasma membrane, and fuse with plasma membrane (fertilization) to produce a zygote. Following fertilization, the ZP is modified by cortical granule components such that acrosome-intact and acrosome-reacted sperm are unable to bind to fertilized eggs. Here we review some of the evidence that bears directly on the involvement of two mouse ZP proteins, mZP2 and mZP3, as receptors for binding of mouse sperm to unfertilized eggs and address some contentious issues surrounding this important initial step in the process of mammalian fertilization., Competing Interests: CONFLICT OF INTEREST STATEMENT The authors declare that they have no disclosures.

Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex. • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. [ABSTRACT FROM AUTHOR]

The sea urchin Arbacia lixula coexist with Paracentrotus lividus in the Mediterranean, but the two sea urchin species are quite different from each other. Concerning the female gamete, A. lixula eggs are much darker than those of P. lividus due to the characteristic pigmentation. Upon insemination, the fertilization envelope formed by A. lixula eggs is remarkably thinner than that of P. livius eggs, which implies that the cortical organization of the eggs in the two species may be quite different. In this communication, we examined the phenotypic plasticity of A. lixula eggs in the changing osmolality. The plasma membrane, cortical actin cytoskeleton and vesicles are extensively altered in the eggs exposed to 40% seawater for 15 min. When fertilized, the Ca2+ response in these eggs was significantly compromised and the sperm often failed to enter the eggs. Remarkably, the pattern of the Ca2+ response was restored when these eggs were transferring back to the natural seawater before fertilization, while the actin cytoskeleton partially reverted to the original state. Nonetheless, these eggs restored in seawater failed to regain the innate sperm receptivity that allows only one sperm to enter in natural seawater. Thus, the ability to guide monospermic fertilization is lost by water entry into the eggs, and the eggs incorporated either multiple or no sperm. On the other hand, eggs briefly exposed to hypertonic seawater exhibited no evident morphological anomaly. Nonetheless, the monospermic eggs that experienced a brief exposure (15 min) to hypertonic seawater prior to fertilization in natural seawater displayed a subtly altered sperm-induced Ca2+ response and morpho-functional anomaly around the pluteus stage. Our results suggest that A. lixula eggs attain only a limited extent of cytological plasticity, and that the osmolality shock affects the physical nature of the egg surface which in turn affects the developmental programming. • Osmotic changes alter egg surface topography. • Water entry into unfertilized sea urchin eggs affects the egg cortex structure. • Hypotonic and hypertonic treatment cause alteration of the sperm-induced Ca2+ response. • Salinity changes affect embryogenesis. [ABSTRACT FROM AUTHOR]

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

Fertilization by multiple sperm leads to lethal chromosomal number abnormalities, failed embryo development, and miscarriage. In some vertebrate and invertebrate eggs, the so-called cortical reaction contributes to their activation and prevents polyspermy during fertilization. This process involves biogenesis, redistribution, and subsequent accumulation of cortical granules (CGs) at the female gamete cortex during oogenesis. CGs are oocyte- and egg-specific secretory vesicles whose content is discharged during fertilization to block polyspermy. Here, we summarize the molecular mechanisms controlling critical aspects of CG biology prior to and after the gametes interaction. This allows to block polyspermy and provide protection to the developing embryo. We also examine how CGs form and are spatially redistributed during oogenesis. During egg activation, CG exocytosis (CGE) and content release are triggered by increases in intracellular calcium and relies on the function of maternally-loaded proteins. We also discuss how mutations in these factors impact CG dynamics, providing unprecedented models to investigate the genetic program executing fertilization. We further explore the phylogenetic distribution of maternal proteins and signaling pathways contributing to CGE and egg activation. We conclude that many important biological questions and genotype-phenotype relationships during fertilization remain unresolved, and therefore, novel molecular players of CG biology need to be discovered. Future functional and image-based studies are expected to elucidate the identity of genetic candidates and components of the molecular machinery involved in the egg activation. This, will open new therapeutic avenues for treating infertility in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rojas, Hinostroza, Vergara, Pinto-Borguero, Aguilera, Fuentes and Carvacho.)