Physiological Polyspermy Research Articles

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians.

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.

Oocyte-sperm interactions in the fertilization of birds

A thorough understanding of the mechanisms leading to the interaction between the sperm and the ovum in the process of fertilization in birds can facilitate more effective programming and control of the reproduction of these animals in breeding farms. In addition, it may allow the introduction of extracorporeal fertilization techniques, which may be important in the creation of transgenic animals and the reproduction of endangered species. In birds, the process of fertilization is not well known. It is conditioned by a series of interactions between mature reproductive cells. Oocytes are formed in the ovarian follicles of the left ovary. After ovulation, an ovum in the metaphase of the second meiotic division enters the oviduct along with the inner perivitelline layer (IPVL). It gets fertilized in this infundibulum. Male gametes are formed in paired testes located in the abdominal cavity. Sperm cells in the female reproductive tract do not require capacitation and are already fully capable of fertilization. As a result of internal insemination, male reproductive cells enter the oviduct. In this organ, they are selected and stored in the primary and secondary sperm storage tubules of the mucous membrane. They are released in batches shortly before ovulation. After reaching the oocyte, the sperm binds to the IPVL. This induces an acrosomal reaction that allows the male reproductive cells to penetrate to the surface of the oocyte, especially at the germinal pole. Next, as a result of physiological polyspermy, many sperm cells reach the ooplasm where they form haploid male pronucleus. This phenomenon is necessary to activate an polylecithal egg and produce a haploid female pronucleus. In the final stage, the female pronucleus merges with the single male pronucleus, which leads to the formation of a diploid zygote. The excess male pronuclei present in ooplasm are broken down by endonucleases (DNases). Understanding the mechanisms leading to the interaction between sperm and oocyte in birds may allow for more accurate programming and breeding of these animals in poultry farms and the introduction of extracorporeal fertilization techniques. In addition, it could be useful for the reproduction of endangered bird species

Fertilization 2: Polyspermic Fertilization.

During fertilization in animals, a haploid egg nucleus fuses with a haploid sperm nucleus to restore the diploid genome. In most animals including mammals, echinoderms, and teleostei, the penetration of only one sperm into an egg is ensured at fertilization because the entry of two or more sperm is prevented by polyspermy block systems in these eggs. On the other hand, several animals such as birds, reptiles, and most urodele amphibians exhibit physiological polyspermy, in which the entry of several sperm into one egg is permitted. However, in these polyspermic eggs, only one sperm nucleus is involved in zygotic formation with a female nucleus, thereby avoiding syngamy with multiple sperm nuclei. In the chicken, 20-60 sperm are generally found within the egg cytoplasm at fertilization and this number is markedly higher than that of other polyspermic species; however, avian-specific events such as the degeneration and mitosis of supernumerary sperm nuclei during early embryo development allow a polyspermic egg to develop normally. This chapter describes current knowledge on polyspermy-related events in avian eggs during fertilization, and is characterized by a comparison to the fertilization modes of other vertebrates. The close relationship between sperm numbers and egg sizes, and the movement of supernumerary sperm nuclei towards the periphery of the egg cytoplasm and their degeneration are summarized. The molecular mechanisms by which polyspermy initiates egg activation to start embryo development are also discussed.

Micropyle number is associated with elevated female promiscuity in Lepidoptera.

In the majority of insects, sperm fertilize the egg via a narrow canal through the outer chorion called the micropyle. Despite having this one primary function, there is considerable unexplained variation in the location, arrangement and number of micropyles within and between species. Here, we examined the relationship between micropyle number and female mating pattern through a comparative analysis across Lepidoptera. Three functional hypotheses could explain profound micropylar variation: (i) increasing micropyle number reduces the risk of infertility through sperm limitation in species that mate infrequently; (ii) decreasing micropyle number reduces the risk of pathological polyspermy in species that mate more frequently; and (iii) increasing micropyle number allows females to exert greater control over fertilization within the context of post-copulatory sexual selection, which will be more intense in promiscuous species. Micropyle number was positively related to the degree of female promiscuity as measured by spermatophore count, regardless of phylogenetic signal, supporting the hypothesis that micropyle number is shaped by post-copulatory sexual selection. We discuss this finding in the context of cryptic female choice, sperm limitation and physiological polyspermy.

Sperm-Egg Interaction during Fertilization in Birds.

Fertilization in animals that employ sexual reproduction is an indispensable event for the production of the next generation. A significant advancement in our understanding of the molecular mechanisms of sperm-egg interaction in mammalian species was achieved in the last few decades. However, the same level of knowledge has not been accumulated for birds because of egg size and the difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. In this review, we summarize the current understanding of sperm-egg interaction mechanism during fertilization in birds, especially focusing on sperm-egg binding, sperm acrosome reaction and the authentic sperm protease required for the hole formation on the perivitelline membrane. We explain that the zona pellucida proteins (ZP1 and ZP3) in the perivitelline membrane play important roles in sperm-egg binding, induction of the acrosome reaction as well as sperm penetration by digestion of sperm protease. We anticipate that a deeper understanding of avian fertilization will open up new avenues to create powerful tools for a myriad of applications in the poultry industries including the production of transgenic and cloned birds.

The birth of quail chicks after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been successfully used to produce offspring in several mammalian species including humans. However, ICSI has not been successful in birds because of the size of the egg and difficulty in mimicking the physiological polyspermy that takes place during normal fertilization. Microsurgical injection of 20 or more spermatozoa into an egg is detrimental to its survival. Here, we report that injection of a single spermatozoon with a small volume of sperm extract (SE) or its components led to the development and birth of healthy quail chicks. SE contains three factors - phospholipase Cζ (PLCZ), aconitate hydratase (AH) and citrate synthase (CS) - all of which are essential for full egg activation and subsequent embryonic development. PLCZ induces an immediate, transient Ca(2+) rise required for the resumption of meiosis. AH and CS are required for long-lasting, spiral-like Ca(2+) oscillations within the activated egg, which are essential for cell cycle progression in early embryos. We also found that co-injection of cRNAs encoding PLCZ, AH and CS support the full development of ICSI-generated zygotes without the use of SE. These findings will aid our understanding of the mechanism of avian fertilization and embryo development, as well as assisting in the manipulation of the avian genome and the production of transgenic and cloned birds.

Egg activation in physiological polyspermy

Fertilization is indispensable not only for restoring diploid genomes but also for the initiation of early embryonic cell cycles in sexual reproduction. While most animals exhibit monospermy, which is ensured by polyspermy blocks to prevent the entry of extra sperm into the egg at fertilization, several animals exhibit physiological polyspermy, in which the entry of several sperm is permitted but only one sperm nucleus participates in the formation of a zygote nucleus. Polyspermy requires that the sperm transmit the egg activation signal more slowly, thus allowing the egg to accept several sperm. An increase in intracellular Ca(2+) concentration induced by the fertilizing sperm is both necessary and sufficient for egg activation in polyspermy. Multiple small Ca(2+) waves induced by several fertilizing sperm result in a long-lasting Ca(2+) rise, which is a characteristic of polyspermic amphibian eggs. We introduced a novel soluble sperm factor for egg activation, sperm-specific citrate synthase, into polyspermic newt eggs to cause Ca(2+) waves. Citrate synthase may perform dual functions: as an enzyme in mitochondria and as a Ca(2+)-inducing factor in egg cytoplasm. We also discuss the close relationship between the mode of fertilization and the Ca(2+) rise at egg activation and consider changes in this process through evolution in vertebrates.

Assembly of the Inner Perivitelline Layer, a Homolog of the Mammalian Zona Pellucida: An Immunohistochemical and Ultrastructural Study

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Coturnix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotemporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development.

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: Its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca2+ increase occurs as a Ca2+ wave at each sperm entry site in the polyspermic egg. Some Ca2+ waves are preceded by a transient spike-like Ca2+ increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca2+ wave was induced by a sperm factor derived from sperm cytoplasm after sperm–egg membrane fusion. The Ca2+ increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca2+ store for the Ca2+ wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.

Adaptive Evolution in an Avian Reproductive Protein: ZP3

Proteins involved in reproduction appear to be evolving adaptively across taxa. This rapid evolution is thought to be the result of forces involved in sexual selection. One of the most often suggested of these forces is sexual conflict involving sperm competition and polyspermy avoidance. Bird species offer a unique opportunity to test this hypothesis since the avian egg coat tolerates physiological polyspermy, or the penetration of multiple sperm during fertilization, without negative effects on later development. Despite this, and the extensive amount of data gathered on sexual selection in birds, there are limited studies on the patterns of evolution of avian reproductive proteins. Here we present an analysis of the pattern of evolution of Zona Pellucida 3 (ZP3), a protein present on the avian egg coat. We found that, across several galliform and a single anseriform species, ZP3 appears to be diverging by positive adaptive evolution. In an exploratory analysis of portions of the gene in Callipepla californica we also found evidence of a selective sweep at the putative sperm binding region of the protein. In sum, ZP3 in birds, like reproductive proteins in other species, appears to be adaptively evolving. This result suggests that polyspermy avoidance is not sufficient to explain positive Darwinian selection in reproductive proteins across taxonomic groups. Clearly, the inclusion of bird species in the study of reproductive proteins across taxa promises to add greatly to the discussion of the factors driving the widespread phenomenon of adaptive evolution in reproductive proteins.

Detection of deoxyribonuclease I and II activities in Japanese quail oocytes

Birds exhibit physiological polyspermy, i.e. numerous spermatozoa enter the germinal disc of an oocyte and form pronuclei during fertilisation. However, only one of them unites with the female pronucleus to form a zygote nucleus; the supernumerary spermatozoal nuclei degenerate at the early cleavage stages. To establish a factor responsible for spermatozoal degeneration, the presence of DNase activity was studied in vitro in extracts of Japanese quail oocytes using lambda DNA/HindIII as a substrate. The experimental conditions were designed to reveal the presence of either DNase I or DNase II activities, separately. Degradation of the substrate DNA was evaluated by electrophoresis on agarose gels stained with ethidium bromide. High activities of DNase I and DNase II were found in the germinal discs of the largest vitellogenic oocytes. DNase I activity was estimated to be about 3 x 10(-3) Kunitz units and DNase II about 4 x 10(-2) Kunitz units per germinal disc. DNase I activity in an oocyte seems to increase during oogenesis since DNA degradation by the extracts from the germinal discs of the largest vitellogenic oocytes was much higher than by those from previtellogenic and small vitellogenic oocytes. The presence of high DNase I and II activities in the largest vitellogenic oocytes would point to their role in degradation of DNA from supernumerary spermatozoa entering the ovum during polyspermic fertilisation in birds. The enzymes could be a factor, or one of the factors, in the late block to polyspermy in the cytoplasm of avian eggs. It is suggested here that the DNase activities might also be responsible for poor efficiency in obtaining transgenic birds by microinjection of exogenous DNA into the fertilised chick ovum.

Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2 O.

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg-activating agents, such as pricking and Ca2+ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non-amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D2 O (that induces microtubule polymerization) and 6-DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster. The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca2+ ions had been chelated by BAPTA could also be activated by either cycloheximide or D2 O. Cycloheximide- as well as 6-DMAP-induced egg activations were not inhibited by nocodazole, a microtubule-depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short-lived proteins acts as an egg activation mechanism, downstream of the site of Ca2+ action and independently of microtubule polymerization.

Numbers of Spermatozoa Attached to and Penetrating Perivitelline Layers of Japanese Quail Eggs

In birds and some other taxa (e.g. urodele amphibians), several spermatozoa typically penetrate the ovum wall at the time of fertilization (referred to as physiological polyspermy) even though the female nucleus fuses with only a single spermatozoan nucleus (Harper 1904, Elinson 1986, Perry 1987). In birds, fertilization takes place in the infundibulum within 15 to 30 min of the ovum being shed from the ovary, at which point one or more spermatozoa penetrate the inner layer of the perivitelline membrane of the ovum (Olsen and Neher 1948). The numbers of spermatozoa in the infundibulum at the time of fertilization can be estimated by counting those trapped on the outer layer of the perivitelline membrane, which is laid down on the ovum soon after fertilization (Gilbert 1971, Wishart 1987). However, the proportion of spermatozoa that undergo the acrosome reaction and penetrate the inner perivitelline layer is not well known (Howarth 1984). The aim of this paper is to describe a simple technique that permits the identification of both: (1) holes caused by the penetration of spermatozoa into the inner perivitelline membrane; and (2) spermatozoa that have not undergone the acrosome reaction and are still attached to the outer perivitelline layer. These two simple techniques open up new possibilities for exploring the dynamics of sperm utilization and sperm competition in birds (Birkhead and M0ller 1992). Methods.-We used fertile and infertile eggs laid within the previous 48 h from naturally mated Japanese Quail (Coturnix japonica). Eggs were opened with scissors and the yolk and albumen separated. The yolk was placed in a petri dish with the germinal disc uppermost. A piece of the perivitelline layer approximately 1 x 1 cm around the germinal disc (diameter = 3.75 mm) was removed with scissors and fine forceps. The piece of membrane was gently washed by shaking in phosphate buffered saline for a few seconds. A second piece of the perivitelline layer (1 x 1 cm) from the same egg, but away from the germinal disc, was also removed and washed. The two pieces were then placed on a microscope slide side by side and laid out flat. We used Wishart's (1987) technique for identifying sperm; this involved placing a drop of fluorescent Hoescht dye 33342 onto the perivitelline tissue and covering with a cover slip. Tissue samples were examined using a transmission microscope. Holes were visible using dark-field optics at a magnification of x 100 (i.e. x 10 objective; Fig. la). Spermatozoa were visible under ultraviolet light at a magnification of x 250 or x 400 ( x 25 or x 40 objective; Fig. lb). The method we used for visualizing holes differs from that of other authors. Bramwell and Howarth (1992b) used Schiff's reagent, and G. J. Wishart (pers. comm.) used a lectin fluorochrome to stain the perivitelline layer in order to increase the contrast between the holes and the layer. However, we found that holes were clearly visible without any stain if we simply used dark-field optics. Moreover, we could use the DNA-specific Hoescht dye 33342 to stain spermatozoa, so that both holes and spermatozoa could be counted from the same piece of perivitelline layer, under dark-field and fluorescence optics, respectively. We examined a single egg laid by each of 15 females, recording the number of holes and spermatozoa over the area of the germinal disc and away from the germinal disc. Counts of holes were made for fields of known area (for holes 3.08 mm2; for spermatozoa 0.48 mm2) and converted to densities per square millimeter. We counted 10 nonoverlapping fields per germinal disc and 30 random fields away from the germinal disc. Densities of spermatozoa and holes were used to estimate the total numbers of spermatozoa and holes over the entire ovum. Each hole in the inner perivitelline layer was assumed to correspond to a single spermatozoa either undergoing the acrosome reaction or undergoing proteolytic activity (see below and Birkhead et al. 1994) and penetrating the layer. Spermatozoa nuclei on the outer perivitelline layer were those that had not undergone the acrosome reaction or proteolytic activity. The surface area (mm2) of the ovum (994 mm2) was calculated using the formula 47rr2 after measuring the diameter of 10 hard-boiled yolks to obtain a mean radius (8.895 mm). Densities are per square millimeter (x ? SE). Results.-The mean densities of both spermatozoa and holes were significantly higher in the germinal disc area than elsewhere on the ovum. The density

Intracellular free Ca2+ changes during physiological polyspermy in amphibian eggs

We have made the first measurements of intracellular free calcium activity ([Ca2+]i) in urodele eggs during physiological polyspermic fertilization. Jellied eggs of the urodele amphibian Pleurodeles waltlii were impaled with intracellular Ca(2+)-selective microelectrodes and inseminated under various conditions of sperm:egg ratio to obtain various degrees of polyspermy. In 17 out of 45 cases the egg [Ca2+]i level (0.41 microM) showed no variation following fertilization. In 28 other cases, however, the egg displayed a slow increase in [Ca2+]i of 0.15 microM, starting around 15 minutes after fertilization and reaching a plateau level around 10 minutes later. The amplitude of the fertilization-associated increase in [Ca2+]i was found to be independent of the number of sperm interacting with the egg surface. Measurements with two Ca(2+)-microelectrodes impaled in single eggs showed that the increase in [Ca2+]i did not simultaneously occur at distinct places within the egg cortex and, in some cases, could be detected by only one of the two microelectrodes. This latter observation, as well as the absence of [Ca2+]i change at fertilization in some experiments, strongly suggested that each sperm interacting with the egg might, at various places, trigger a localized, non-propagating change in [Ca2+]i. Experiments in which eggs were locally inseminated, using a micropipette directed towards the site of impalement of one of the two Ca(2+)-microelectrodes, clearly established that [Ca2+]i changes, although incapable of propagating over the entire egg cortex, might nevertheless travel very slowly over short distances, their amplitude vanishing rapidly as they propagate from around the sites of sperm entry.(ABSTRACT TRUNCATED AT 250 WORDS)

A long-lasting electrically mediated block, due to the egg membrane hyperpolarization at fertilization, ensures physiological monospermy in eggs of the crab Maia squinado

In response to fertilization, the membrane potential ( E m) of the crab egg hyperpolarizes from about −50 mV to about −80 mV in 400 msec. To establish whether this fast hyperpolarization is correlated with physiological polyspermy or conversely mediates an electrical block to polyspermy, we examined the morphological and electrophysiological characteristics of eggs from the crab Maia squinado. Fertilized naturally spawned eggs were found to be physiologically monospermic and their average E m was constant at −77 ± 0.5 mV. To examine a possible electrical block ensuring this monospermy, unfertilized eggs were voltage clamped at various E m values ranging from +20 to −90 mV, inseminated, and examined morphologically. All eggs clamped at +20 to −65 mV responded by developing a fertilization current, I f. It consisted of an outwardly directed K + current in one or several steps, each caused by a single spermatozoon interacting with the egg membrane. The percentage of eggs clamped at values more negative than −65 mV, which responded at insemination by developing an I f, decreased and dropped to 0 at −80 mV. This indicated that the membrane processes occurring during the contact between gametes and eliciting an electrical response by the egg membrane are voltage dependent. Further, the spermatozoon never penetrated into eggs voltage clamped at a E m between +20 and −60 mV and at voltages more negative than −75 mV. E m values between −65 and −75 mV were required for spermatozoon incorporation into the egg, indicating that sperm entry is also voltage dependent. It is proposed that the hyperpolarization of the egg membrane in response to fertilization constitutes a long-lasting electrical block to polyspermy in crab eggs.

Fertilization in Amphibians: The Ancestry of the Block to Polyspermy

Publisher Summary Amphibians exhibit both styles of fertilization: monospermy and polyspermy. Monospermy is confined to tailless frogs and toads that comprise the order Anura, while physiological polyspermy is found in the tailed newts and salamanders of the order Urodela. This chapter reviews the process of fertilization in amphibians, particularly as it relates to polyspermy. The chapter discusses ways to explore the nature of the cytological ancestry. Amphibians exhibit a great diversity of reproductive patterns. This diversity makes the amphibians ideal animals for examining the cellular evolution of developmental processes. Fertilization in anurans and urodeles is so different that it represents an important character distinguishing the orders. Comparison between fertilization in anurans and urodeles shows a mirror image dichotomy with respect to polyspermy. The anuran egg has a fast and slow block to polyspermy, and it has no protection when more than one sperm enters. On the other hand, the urodele egg lacks both external blocks to polyspermy but can deal with accessory nuclei within the egg. The arrangement of components in the membranes and in the cortical granules is considered important for the polyspermy-blocking reactions.

The membrane potential changes of amphibian eggs during species- and cross-fertilization

The mature eggs of the newt Cynops pyrrhogaster, which exhibit physiological polyspermy, had a resting membrane potential of about −16 mV, an input resistance of about 64 MΩ, and a time constant of 5.7 sec. Upon insemination with homologous sperm, the eggs generated several small hyperpolarizations (2–10 mV) lasting for 30–90 sec at 2- to 3-min intervals. The number of these hyperpolarizations in each egg corresponded well to the number of the sperm in those eggs that were observed in cytological sections. When the eggs of the frog Xenopus laevis were inseminated with the Cynops sperm, polyspermic fertilization occurred instead of monospermy with the homologous sperm. In these cross-fertilized eggs, the cortical reaction occurred normally but in an apparent delay when compared with that during the self species fertilization. Upon insemination with the Cynops sperm, the Xenopus eggs having a resting membrane potential of −10 mV underwent a gradual hyperpolarization to −35 mV, which was never observed during the self species fertilization. The eggs then generated a rapid depolarization amounting to +30 mV. The experiments on various ionic conditions indicate that the former hyperpolarization and the latter depolarization are mediated by different ionic permeability of the egg plasma membrane.

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

AbstractThe electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K+ dependent resting potentials, Em, of −61 ∓ 1 mV, n=19, and −67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of −40 ± 1.5 mV, n=19, that was k+ and Cl− dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl− and the Em attained–41 ± 1 mV, n=49 and −34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K+ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.

Fertilization of amphibian eggs: A comparison of electrical responses between anurans and urodeles

In Pleurodeles waltl and Ambystoma mexicanum, which exhibit physiological polyspermy, the membrane potential in most eggs did not change in any consistent pattern during 45 min after fertilization; in some cases, a slow hyperpolarization began 5 to 15 min after insemination and continued for 10–15 min. These eggs then slowly depolarized, reaching a stable value of −10 to +10 mV, about 45 min after fertilization. Membranes of eggs activated by A23187 or by electrical stimulus showed a similar behavior. The diversity of responses does not correlate with the number of sperm fusing with the egg. Holding the membrane potential at a constant value between −40 and +40 mV during insemination did not prevent fertilization nor delay sperm-egg interactions. The fertilization or activation potential of Rana temporaria eggs consists of a rapid (1 sec) depolarization accompanied by a sudden decrease in membrane resistance. The activation potential can be triggered by A23187 and by calcium iontophoresis; its amplitude depends on the (Cl −) 0 and to a lesser extent on the (Na +) 0. Fertilization was prevented when the membrane potential was clamped above +15 mV. However, slowing the rise time (5 to 8 sec instead of 1 sec) and reducing the amplitude (10–20 mV instead of 40–60 mV) of the fertilization potential, both by injecting negative current, never induced polyspermy.

DNA synthesis after polyspermic fertilization in the axolotl

Cytological and autoradiographic studies were done to investigate the cytoplasmic control of DNA synthesis under conditions of physiological polyspermy. The DNA synthetic phases of the egg, principal sperm and accessory sperm nucleic were determined and correlated with nuclear morphology and develpmental fate. Results show that accessory sperm nuclei undergo morphological transition to pronuclei. Their DNA synthetic phase is the same as that of the principal sperm nucleus. Hence accessory sperm nuclei are capable of initiating and completing DNA replication before any cytological evidence of their degeneration is observed.