Yasuhiro Iwao

Molecular Identification and Characterization of Xenopus Egg Uroplakin III, an Egg Raft-associated Transmembrane Protein That Is Tyrosine-phosphorylated upon Fertilization

Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.

Participation of oviducal pars recta secretions in inducing the acrosome reaction and release of vitelline coat lysin in fertilizing toad sperm

Jellyless coelomic eggs of the toad, Bufo bufo japonicus, are not fertilizable when inseminated in jelly components that assure fertilization of dejellied uterine eggs. However, when coelomic eggs were pretreated with an extract from the uppermost portion of oviduct (pars recta) and inseminated in jelly, a high frequency of fertilization was obtained. This activity was observed only in the pars recta extract (PRE) prepared from pituitary-injected, ovulating females. Treatment of coelomic eggs with the PRE followed by addition of sperm without jelly resulted in lysis of the vitelline coat (VC). Likewise, the supernatant of the PRE preincubated with sperm had a strong VC lytic activity. The Triton X-100 extract of sperm preincubated with the PRE exhibited less than 20% of the proteolytic activity in extracts of control, untreated sperm. Electron microscopy of sperm revealed that the PRE treatment induces a specific loss of the acrosomal cap. Electron microscopically, distinct filament bundles of the VC in coelomic eggs became less conspicuous, after eggs passed through the pars recta or were treated with the PRE. Epithelial cells in the pars recta contained unique nonciliated cells that possess electron-dense granules distinct from those in jelly-secreting cells in lower oviducal portions. The number of these granules in each cell increased significantly upon injection of pituitary hormone into females. Immunohistochemical observations proved that the specific antigen(s) localized in nonciliated cells in the pars recta is not detected in the VC of coelomic eggs, but is deposited in the VC during passage of eggs through the pars recta. It is concluded that the substance(s) secreted from the pars recta cells is deposited in the VC, and induces the acrosome reaction and release of VC lysin in sperm to ensure that they make a successful fusion with the egg plasma membrane.

Evidence that the voltage-dependent component in the fertilization process is contributed by the sperm

To investigate the mechanisms that account for the voltage dependence of fertilization and provide an electrical block to polyspermy, we studied cross-fertilizations between three species of amphibians having different degrees of voltage dependence. Anurans, such as the toad Bufo japonicus, as well as the primitive urodele Hynobius nebulosus, have voltage-dependent fertilization; other urodeles, such as Cynops pyrrhogaster, have voltage-independent fertilization (Y. Iwao, 1989, Dev. Biol. 134, 438-445). Entry of Hynobius sperm into Cynops eggs was blocked by clamping the eggs membrane potential at +40 mV, as is the case for fertilization of Hynobius eggs with Hynobius sperm, but not for fertilization of Cynops eggs with Cynops sperm. Therefore, fertilization was voltage dependent in an experimental condition where only the sperm could be contributing this characteristic. The voltage-dependent properties of fertilization between Bufo eggs and Hynobius sperm were also characteristic of the sperm species; fertilization was blocked at +50 mV as in Hynobius fertilization, but not at +20 mV as in Bufo fertilization. These results support the conclusion that the voltage dependence of fertilization results from a component contributed by the sperm.

Mechanisms of Egg Activation and Polyspermy Block in Amphibians and Comparative Aspects with Fertilization in Other Vertebrates

Abstract For precise temporal activation of the egg during amphibian fertilization, the sperm must provide a signal for egg activation at the time of membrane binding or fusion between sperm and eggs. A fertilizing sperm causes a Ca2+ wave which is both necessary and sufficient for egg activation at amphibian fertilization. The Ca2+ wave seems to be mediated by IP3-receptors on the endoplasmic reticulum and by IP3 produced by hydrolysis of PLC activated by a Src-related protein tyrosine kinase (Xyk) in Xenopus eggs. We have proposed three different hypotheses for initiation of egg activation in amphibian eggs: the Ca2+-influx model, the membrane receptor model, and the soluble factor model. The membrane receptor model and the soluble factor model seems to be applied to the monospermic Xenopus fertilization and the physiologically polyspermic Cynops fertilization, respectively. The Ca2+ wave at egg activation induces a positive fertilization potential which prevents entry of a second sperm in fertilization of monospermic species. In physiologically polyspermic urodele eggs, several sperm enter the egg at normal fertilization, but only one sperm nucleus with a centrosome participates in the embryonic development. The degeneration of accessory sperm nuclei is closely involved in differential distributions of both γ-tubulin and cyclin B in the egg cytoplasm, which causes developing a larger sperm aster and earlier entry into M phase in a zygote nucleus, respectively. We have discussed the molecular mechanisms of egg activation and polyspermy blocks in amphibians and make some comparisons with other vertebrates, such as fishes and mammals.

An electrically mediated block to polyspermy in the primitive urodele Hynobius nebulosus and phylogenetic comparison with other amphibians.

At fertilization, the egg of the primitive urodele, Hynobius nebulosus, produced a fertilization potential which rose from -12 to +47 mV. A similar activation potential was elicited by pricking with a needle, by applying A23187, or by electric shock. The potential change was mediated by an increased permeability to Cl-. Clamping the eggs membrane potential at +40 mV blocked fertilization, while clamping at +20 mV induced polyspermy. These results indicated the occurrence of an electrical polyspermy block, typical of anurans, but atypical of urodeles. Furthermore, Hynobius eggs fertilized by natural mating incorporated only one sperm nucleus, and experimentally polyspermic eggs underwent multipolar division. Accessory sperm did not degenerate in the egg cytoplasm, indicating lack of an intracellular polyspermy block. By comparison, fertilization of Bufo japonicus (anuran) was also voltage dependent, whereas that of Cynops pyrrhogaster (urodele) was voltage independent. Thus polyspermy prevention mechanisms in Hynobius closely resemble those of anuran amphibians and differ from those of higher urodeles.

Control of sperm nuclear behavior in physiologically polyspermic newt eggs: Possible involvement of MPF

We have studied the mechanism controlling the behavior of accessory sperm nuclei in physiologically polyspermic eggs of the newt, Cynops pyrrhogaster. Our approach was to identify cytoplasmic components which would prevent the usual degeneration of accessory sperm nuclei. Injection of cytoplasm from unfertilized eggs, but not fertilized ones, induced multipolar cleavage in polyspermically fertilized eggs as well as centrosome separation and formation of extra bipolar spindles in accessory sperm nuclei. Cytosols extracted from unfertilized Cynops or Xenopus eggs also were active in inducing multipolar cleavage, as were germinal vesicle materials from oocytes of the frogs Xenopus or Rana or of Cynops. In all of these cases, the nuclear cycle as well as the onset of first cleavage was delayed relative to those in control eggs. In contrast, injection of an extract with maturation-promoting factor (MPF) activity, prepared from unfertilized Xenopus eggs, induced precocious and multipolar cleavage when injected into fertilized Cynops eggs. Injection of the MPF-containing extract caused acceleration of the nuclear cycle as well as formation of extra bipolar spindles by the accessory sperm nuclei. These results suggest that a local deficiency of MPF may lead to the degeneration of accessory sperm nuclei in physiologically polyspermic eggs.

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

Abstract 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.

Activation of Xenopus Eggs by an Extract of Cynops Sperm

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.

Fertilization in Amphibians

Fertilization of a female gamete or oocyte by a male gamete or sperm is an indispensable step in sexual reproduction in animals. Since both sperm and oocytes are highly specialized to ensure development of offspring with a diploid genome, many molecules are involved in cell-cell adhesion, membrane fusion, and signaling cascades until the karyogamy between sperm and oocyte nuclei. This chapter focuses on cellular and molecular aspects of fertilization in amphibians, which are one of the best studied animals for fertilization in vertebrates. The mechanisms of polyspermy block to ensure diploid development are discussed in relation to extracellular interactions between the sperm and the oocytes as well as oocyte activation by the sperm.

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs.

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6‐fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37‐fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997.