Yolk platelets impede nuclear expansion in Xenopus embryos.

During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. Nuclear expansion was slower in blastomeres from vegetal hemispheres during a longer interphase than in those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, nuclear expansion and DNA replication became slower than that in normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded the organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion and cell cycle progression in the yolk-rich vegetal blastomeres.

SPERM FACTORS AND EGG ACTIVATION: Divergent sperm factors for egg activation in amphibian fertilization.

Mature amphibian eggs arrested at meiotic metaphase II must undergo activation to initiate embryonic development soon after fertilization. Fertilizing sperm provide eggs with a signal that induces egg activation, and an increase in intracellular Ca2+ concentration in the egg cytoplasm (a Ca2+ rise) is the most important signal for this initiation. The sperm transmits the signal for the Ca2+ rise, known as the sperm factor, which is divergent between anurans and urodeles. In monospermic anurans, the sperm transmits the signal through a receptor on the egg membrane, causing a single rapid Ca2+ rise. Sperm matrix metalloproteinase-2 is a potential candidate for the receptor-mediated sperm factor in anurans. In physiologically polyspermic urodeles, multiple slower Ca2+ rises are caused by a soluble sperm factor (sperm-specific citrate synthase) which is transferred to the egg cytoplasm after sperm-egg fusion. We discuss the molecular mechanisms of egg activation in amphibian fertilization, focusing on recent progress in characterizing these sperm factors and their divergence during the evolution of tetrapod vertebrates.

Sperm MMP-2 is indispensable for fast electrical block to polyspermy at fertilization in Xenopus tropicalis.

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 ΔHPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.

Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

Men and women become infertile with age, but the mechanism of declining male fertility, more specifically, the decrease in in sperm quality, is not well known. Citrate synthase (CS) is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, which directly controls cellular function. Extra-mitochondrial CS (eCS) is produced and abundant in the sperm head; however, its role in male fertility is unknown. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in egg fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped with age. Metabolomic analysis of aged sperm revealed that the loss of eCS enhances TCA cycle in the mitochondria with age, presumably leading to depletion of extra-mitochondrial citrate. The data suggest that eCS suppresses age-dependent male infertility, providing insights into the decline of male fertility with age.

Correction: Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

The electrical block to polyspermy induced by an intracellular Ca2+ increase at fertilization of the clawed frogs, Xenopus laevis and Xenopus tropicalis.

Polyspermy blocking, to ensure monospermic fertilization, is necessary for normal diploid development in most animals. We have demonstrated here that monospermy in the clawed frog, Xenopus tropicalis, as well as in X. laevis, is ensured by a fast, electrical block to polyspermy on the egg plasma membrane after the entry of the first sperm, which is mediated by the positive-going fertilization potential. An intracellular Ca2+ concentration ([Ca2+ ]i ) at the sperm entry site was propagated as a Ca2+ wave over the whole egg cytoplasm. In the X. tropicalis eggs fertilized in 10% Steinberg's solution, the positive-going fertilization potential of +27 mV was generated by opening of Ca2+ -activated Cl- -channels (CaCCs). The fertilization was completely inhibited when the egg's membrane potential was clamped at +10 mV and 0 mV in X. tropicalis and X. laevis, respectively. In X. tropicalis, a small number of eggs were fertilized at 0 mV. In the eggs whose membrane potential was clamped below -10 mV, a large increase in inward current, the fertilization current, was recorded and allowed polyspermy to occur. A small initial step-like current (IS current) was observed at the beginning of the increase in the fertilization current. As the IS current was elicited soon after a small increase in [Ca2+ ]i , this is probably mediated by the opening of CaCCs. This study not only characterized the fast and electrical polyspermy in X. tropicalis, but also explained that the initial phase of [Ca2+ ]i increase causes IS current during the early phase of egg activation of Xenopus fertilization.

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 Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) 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.

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.

Role of the PI3K-TOR-S6K pathway in the onset of cell cycle elongation during Xenopus early embryogenesis.

In the early embryogenesis of the frog, Xenopus laevis, cells proliferate by rapid and synchronous divisions, followed by cell cycle elongation and prolongation of the S phases, and then the appearance of the G2 and G1 phases after the midblastula transition (MBT). The beginning of cell cycle elongation was thought to depend on an increase in the nucleo-cytoplasmic (N/C) ratio in blastomeres and a decrease in cortical cytoplasmic factors necessary for cell cycle progression, although these factors are unknown. In the present study, we demonstrated that a regulatory subunit of PI3K (p85α) was localized in the cortical cytoplasm of the blastomere during the MBT. When the embryos were treated with a PI3K inhibitor, LY294002, or a TOR inhibitor, rapamycin, cell cycle elongation was initiated before the MBT. In addition, the inhibition of S6K expression by antisense morpholino oligo enhanced the initiation of cell cycle elongation. In contrast, the activation of PI3K-TOR by Rheb-S16H expression delayed the initiation of cell cycle elongation. These results indicate that a decrease in translational activity dependent on the PI3K-TOR-S6K pathway causes the initiation of cell cycle elongation at the onset of the MBT.

Evidence for true fall-mating in Japanese newt Cynops pyrrhogaster.

The mating season of Japanese newt Cynops pyrrhogaster is generally thought to occur once a year in spring to early summer, during the months of April to June, as in many other Japanese amphibians. However, in fall, from September to October, we often observed breeding colored males demonstrating a mating behavior with females in the field. In this study, in order to identify their true mating season, we anatomically and histologically investigated the annual maturation cycle of gonads and reproductive organs, including cloacal spermathecae in females, and, using a molecular marker, identified the seasonal origins of sperm, which are released in spring to perform insemination. We found that, in fall, ovaries are somewhat immature, while the testes were mature and the sperm already stored in the deferent ducts. Females stored a significant amount of sperm in around 80% of the spermatechae examined in October and 100% in December. When artificially ovulated in March before contact with male partners after hibernation, the females spawned fertilized eggs and these developed normally. Finally, we identified heterozygous genotypes of the visual pigment gene for the two different population types in the embryos, which were derived from a female who established contact with males of the same population in fall and then switched to males from another population until oviposition in spring. We therefore, conclude that the true mating season of this species occurs from fall to early summer, interrupted only by winter, and lasts six months longer (from October to June) than generally believed.

Analysis of a sperm surface molecule that binds to a vitelline envelope component of Xenopus laevis eggs.

To analyze sperm surface molecules involved in sperm-egg envelope binding in Xenopus laevis, heat-solubilized vitelline envelope (VE) dot blotted onto a polyvinylidene difluoride (PVDF) sheet was incubated with a detergent extract of sperm plasma membrane (SP-ML). The membrane components bound to the VE were detected using an antibody library against sperm plasma membrane components, and a hybridoma clone producing a monoclonal antibody (mAb) 16A2A7 was identified. This mAb was used in a Far Western blotting experiment in which VE was separated by electrophoresis, and then transferred to a PVDF strip that was incubated with SP-ML. It was found that SP-ML binds to the VE component gp37 (Xenopus homolog of mammalian ZP1). The antigens reactive to mAb 16A2A7 showed apparent molecular weights of 65-130 and 20-30 kDa, and were distributed relatively evenly over the entire sperm surface. Periodate oxidation revealed that both the pertinent epitope on the sperm surface and the ligands of VE gp37 were sugar moieties. VE gp37 was exposed on the VE surface, and the mAb 16A2A7 dose-dependently inhibited sperm binding to VE. The sperm membrane molecules reactive with mAb 16A2A7 also reacted with mAb 2A3D9, which is known to recognize the glycoprotein SGP in the sperm plasma membrane and is involved in interactions with the egg plasma membrane, indicating that the sperm membrane glycoprotein has a bifunctional role in Xenopus fertilization.

DNA content contributes to nuclear size control in Xenopus laevis.

Cells adapt to drastic changes in genome quantity during evolution and cell division by adjusting the nuclear size to exert genomic functions. However, the mechanism by which DNA content within the nucleus contributes to controlling the nuclear size remains unclear. Here, we experimentally evaluated the effects of DNA content by utilizing cell-free Xenopus egg extracts and imaging of in vivo embryos. Upon manipulation of DNA content while maintaining cytoplasmic effects constant, both plateau size and expansion speed of the nucleus correlated highly with DNA content. We also found that nuclear expansion dynamics was altered when chromatin interaction with the nuclear envelope or chromatin condensation was manipulated while maintaining DNA content constant. Furthermore, excess membrane accumulated on the nuclear surface when the DNA content was low. These results clearly demonstrate that nuclear expansion is determined not only by cytoplasmic membrane supply but also by the physical properties of chromatin, including DNA quantity and chromatin structure within the nucleus, rather than the coding sequences themselves. In controlling the dynamics of nuclear expansion, we propose that chromatin interaction with the nuclear envelope plays a role in transmitting chromatin repulsion forces to the nuclear membrane.

The Sperm-surface glycoprotein, SGP, is necessary for fertilization in the frog, Xenopus laevis.

To identify a molecule involved in sperm-egg plasma membrane binding at fertilization, a monoclonal antibody against a sperm-surface glycoprotein (SGP) was obtained by immunizing mice with a sperm membrane fraction of the frog, Xenopus laevis, followed by screening of the culture supernatants based on their inhibitory activity against fertilization. The fertilization of both jellied and denuded eggs was effectively inhibited by pretreatment of sperm with intact anti-SGP antibody as well as its Fab fragment, indicating that the antibody recognizes a molecule on the sperm's surface that is necessary for fertilization. On Western blots, the anti-SGP antibody recognized large molecules, with molecular masses of 65-150 kDa and minor smaller molecules with masses of 20-28 kDa in the sperm membrane vesicles. SGP was distributed over nearly the entire surface of the sperm, probably as an integral membrane protein in close association with microfilaments. More membrane vesicles containing SGP bound to the surface were found in the animal hemisphere compared with the vegetal hemisphere in unfertilized eggs, but the vesicle-binding was not observed in fertilized eggs. These results indicate that SGP mediates sperm-egg membrane binding and is responsible for the establishment of fertilization in Xenopus.

Differential Emergence of Cortical Granule Breakdown and Electrophysiological Responses during Meiotic Maturation of Toad Oocytes: (activation potential/cortical granule breakdown/ionophore A23187/oocyte maturation).

Oocytes of the toad, Bufo bufo japonicus, at various stages of progesterone-induced maturation were stimulated by pricking or treatment with Ca-ionophore A23187. Upon pricking, oocytes 14 hr after hormone treatment (PHT) underwent sequential activation responses, such as development of an activation potential, cortical granule breakdown (CGBD), and formation of a perivitelline space (PVS), like those of mature oocytes (18 hr PHT). When oocytes were pricked at 14 hr PHT, it took about 10 min for the wave of CGBD to spread over all the oocyte surface, in contrast to the case with mature oocytes in which it took about 150 sec. The rate of spread of CGBD was significantly less in the vegetal hemisphere than in the animal hemisphere in both mature and immature oocytes. Treatment with A23187 (1 µM) for 5 min induced an activation potential, and PVS formation by the oocytes from 10 hr PHT, which was 3-4 hr earlier than the time when these responses could be induced by pricking. Oocytes at 8-9 hr PHT also showed CGBD in response to A23187, but without formation of an activation potential. Several patches of local PVS caused by the non-propagating CGBD were observed in oocytes treated with the ionophore 5-7 hr PHT. When a high concentration (10 µM) of A23187 was employed, CGBD without PVS formation was induced even in oocytes at 0 hr PHT. These results indicate that the responsiveness to a Ca2+ surge that is a prerequisite for both CGBD and genesis of an activation potential is acquired for the repective responses at different stages of oocyte maturation.

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.

A Factor That Interrupts the Cell Cycle at G2 or Prophase is Present in Full-Grown Frog Oocytes: (MPF/cell cycle/oocyte maturation/prophase arrest).

Full-grown amphibian oocytes that had been arrested at meiotic prophase I contained an activity that prevented the cell cycle from progressing beyond a G2 -like stage. Injection of the contents of germinal vesicles (GV-content) or cytoplasm obtained from oocytes of the frog Rana rugosa prevented fertilized eggs of Cynops pyrrhogaster or Bufo japonicus from cleaving. The nuclei in the arrested eggs consisted of thin chromosomes and nucleolus-like particles enclosed within clear nuclear membrane and their volume increased as a function of time after injection. Cycling of maturation-promoting factor (MPF) did not occur in the injected eggs, but DNA synthesis was not disturbed. The injection of exogenous MPF into the eggs induced the reinitiation of the cell cycle with progression to the M phase and subsequent cleavage. Furthermore, the injection into the full-grown oocytes of Bufo inhibited induction of the maturation of oocytes by progesterone. These results demonstrate that a factor that arrests the cell cycle either at a G2 -like stage of mitosis or at prophase in meiosis is present both in the GV and cytoplasm of frog oocytes. We refer to this factor as a G2 -specific cytostatic factor (G2 -CSF). G2 -CSF may play an important role not only in the physiological arrest at prophase I in meiosis, but also in regulation of the G2 /M transition in the cell cycle of early embryonic cells.

Electrical Properties of Toad Oocytes During Maturation and Activation.

The full-grown oocytes of the toad Bufo bufo japonicus, whether in follicular layer or not, had a membrane potential of about -50 mV in De Boer's solution (DB), but underwent a deep hyper-polarization of up to -90 mV when pretreated with Ca, Mg-free EDTA-solution. Regardless of the magnitude of their resting potentials, the defolliculated oocytes exposed to progesterone underwent a gradual depolarization before the germinal vesicle breakdown and retained membrane potential at a level of -10 mV until 18 hr post hormone treatment (PHT), the stage of the second meiotic metaphase. A positive-going activation potential of a magnitude of 70 mV was recorded in the oocytes when pricked at 18 hr PHT as well as in uterine eggs 3-5 min after insemination. A low magnitude of activation potential in response to pricking was recorded in 63% of the oocytes at 13 hr PHT, and premature oocytes exhibiting the activation potential always underwent cortical granule breakdown (CGBD) and perivitelline space formatión. Oocytes where the germinal vesicle had been removed before the hormone treatment exhibited an activation potential and underwent CGBD in response to pricking at 18 hr PHT, whereas those pulse-treated with cycloheximide (10 µg/ml) during the 8-11 hr PHT exhibited neither of these cortical responses. These results indicate that the syntheses of proteins independent of germinal vesicle taking place at 9-11 hr PHT enable the oocytes to undergo cortical responses.

Experiments Pertaining to the Suppression of Accessory Sperm in Fertilized Newt Eggs*: (polyspermy/cleavage/sperm nucleus/newt egg).

In the physiologically polyspermic eggs of the newt, Cynops pyrrhogaster, a number of accessory sperm undergo pronuclear formation along with a concomitant DNA synthesis, but degenerate after zygote nucleus formation. When denuded eggs were divided into two halves at various post-fertilization stages, the andromerogons produced before zygote nucleus formation but not after that stage cleaved at a high frequency. The accessory sperm were unable to participate in the cleavage when they were located in the half of the egg which was connected with the diploid merogon by a cytoplasmic bridge higher than 100 µm in height. The removal of the egg nucleus or the retardation of early post-fertilization nuclear events by treatment with cycloheximide resulted in the induction of multipolar cleavage. Continuous exposure of the fertilized eggs to aphidicolin showed that in the appreciable absence of the DNA synthesis many eggs underwent a first cleavage cytokinesis of a mostly abortive type, but failed to initiate the following cytokinesis at all. Cytological examinations in association with these experiments suggest that the observed suppression of accessory sperm includes the inhibition of centriolar replication under the influence of the zygote nucleus, resulting in the failure of cytasters corporating with nuclear-independent activity of cortical cytoplasm.

Activation of Xenopus Eggs by an Extract of Cynops Sperm: (fertilization/egg-activation/polyspermy block/sperm protease).

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca2+ ions, [Ca2+ ]0 , was reduced to 1.5 µM, but it was enhanced when [Ca2+ ]0 was increased to 340 µM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca2+ ]0 was 340 µM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca2+ ions that results in voltage-insensitive activation of the egg.