Shunichi Miyazaki

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Sperm-specific phospholipase C-ζ (PLCζ) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCζ has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at ∼100 nm Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408–10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCζ activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCζ activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCζ.

Periodic hyperpolarizing responses in hamster and mouse eggs fertilized with mouse sperm

1. The zona‐free hamster egg allows multiple entries of heterologous as well as homologous sperm. The hamster egg inseminated with mouse sperm (M × H egg) showed recurring, transient hyperpolarizing responses (h.r.s) with the peak of ‐70 to ‐80 mV. They were superimposed on a hyperpolarizing shift of the resting potential (h.s.) which gradually reached ‐60 mV in 50 min after insemination.

Development of excitability in embryonic muscle cell membranes in certain tunicates.

During the course of development of muscle cells in certain tunicates, a sign of regenerative membrane response appears in the gastrula stage. In the early tadpole larva, the action potential consists of a spike followed by a plateau. The latter-disappears in fully differentiated cells, conceivably in association with the establishment of delayed rectification.

Calcium and Sodium Contributions to Regenerative Responses in the Embryonic Excitable Cell Membrane

Ionic dependence of regenerative responses of the embryonic cell mnembrane has been studied successively at each stage of development from the unfertilized egg to the differentiated striated muscle in the tadpole larva of the tunicate. The unfertilized egg cell itself showed a type of regenerative response dependent on both sodium and calcium ions, while the spike potentials exclusively dependent on calcium ions were elicited in the differentiated muscle cell.

Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.

1. Synergistic activation of a GTP‐binding protein (G protein) by external serotonin (5‐hydroxytryptamine, 5‐HT) and internally applied guanosine‐5‐O‐(3‐thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM‐5‐HT caused a single HR of 10‐12 s duration and with a delay of 80 s. The critical concentration of 5‐HT to cause an HR was 50 nM. 3. With 10 microM‐5‐HT four to six HRs were often elicited with a delay to the first HR of 8‐30 s. HRs disappeared after prolonged or repeated application of 5‐HT, indicating an apparent desensitization. 4. 5‐HT‐induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3‐9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120‐240 s followed by a series of much smaller HRs. When 5‐HT was applied within 1 min of injection of GTP gamma S. 70 nM‐5‐HT induced a number of large HRs and even 1 nM‐5‐HT could induce HR(s). In contrast, when 5‐HT was applied after the size of GTP gamma S‐induced HRs had declined, as much as 10 microM‐5‐HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5‐HT. 6. GTP gamma S‐induced Ca2+ transients were facilitated when injected in the presence of 5‐HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM‐5‐HT or 4 s in 100 nM‐5‐HT whereas it was 170 s without 5‐HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5‐HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S‐plus‐5‐HT‐potentiated HRs after the first four to five HRs. Sphingosine (2 microM) augmented the series of HRs.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of non-linearity observed in the current—voltage relation of the tunicate embryo

1. In the gastrula of the tunicate (Halocynthia aurantium) the resting potential of the embryonic membrane was about −70 mV in both std ASW and Na‐free ASW.

Electrical excitability in the egg cell membrane of the tunicate

1. With the purpose of studying the differentiation of an excitable membrane, the electrical properties of the tunicate egg, a mosaic egg, was examined by intracellular recording techniques. The species used were Halocynthia aurantium Pallas and H. roretzi Drashe.

Fertilization Potential and Calcium Transients in Mammalian Eggs

The early physiological events at fertilization of eggs involve a change in membrane potential called the fertilization (or activation) potential and a dramatic, transient increase in the intracellular free-calcium ion concentration ([Ca”Ii) (9). These phenomena have been analysed extensively in the sea urchin, and it is known that the fertilization potential is important for fast electrical block of polyspenny and that the increase in [Ca2+Ii causes cortical vesicle exocytosis, which leads to formation of the fertilization membrane and to establishment of permanent block of polyspermy. The increase in [Ca2+Ji, which takes the form of a “Ca wave” due to propagation of release of Ca2+ from intracellular stores, has also been shown to cause increase in 0 2 consumption and activation of NAD kinase in the sea urchin egg, and is thought to be indirectly related to the metabolic activation of inseminated eggs through a still unknown mechanism (9). Reports of studies on these phenomena in mammalian eggs, mainly those of the golden hamster, have appeared only since 1981. In these eggs, the underlying events at fertilization are basically similar to those in the sea urchin, but the patterns of change in membrane potential and increase in [Ca2+Ii are quite different. Here, I review studies on these phenomena, mainly in hamster eggs, and discuss the possible mechanism of signal transduction of sperm-egg interaction causing increases in [ca2+Ii (Ca2+ transients).

Establishment of monolayer culture of pig pancreatic endocrine cells by use of nicotinamide

A method for the isolation and primary monolayer culture of adult pig pancreatic endocrine (PE) cells was established. Cells were dissociated from the pancreas by autodigestion without addition of proteolytic enzymes and separated into distinct bands in a single centrifugation step using Histopaque-1077 (a mixture of polysucrose and sodium diatrizoate). The cells collected from an interfacial fraction were suspended in RPMI 1640 containing 11 mmol/l D-glucose with or without nicotinamide (0, 10, 20, 40 mmol/l), and then placed in culture dishes. Pancreatic cells formed a monolayer while fibroblasts became detached from the bottom of the dish when cultured in the presence of nicotinamide. More than 80% of monolayer-forming cells were stained for insulin, using an enzymatic method, and were identified as B-cells. Morphologically, the PE cells extended multiple processes terminating in growth-cone-like structures, as visualized by both light microscopy and scanning electron microscopy. Insulin secretion in response to glucose stimulation occurred for 35 days of incubation in the RPMI 1640 medium, with or without nicotinamide. Exposure of the cells to nicotinamide for 35 days resulted in a 2-3-fold increase in insulin secretion in response to high glucose stimulus (16.7 mmol/l) compared with low glucose (5.5 mmol/l). Glucose-induced Ca2+ responses were examined in individual cells cultured for 35 days in the presence of 10 mmol/l nicotinamide, using Ca2+ imaging with fura-2. These results indicate that it is possible to prepare pig PE cells in monolayer culture with low fibroblast contamination and to maintain functioning B-cells in vitro for relatively long periods. The present method provides useful preparations for further morphological and physiological studies on the differentiation, growth and regenerative capacity of islet cells.

Intracellular calcium oscillations in mammalian eggs at fertilization.

I am honoured to give a classical perspective on our old paper: Igusa & Miyazaki (1983). n nIn 1981, we first reported that a change in the membrane potential at fertilization, the fertilization potential, in golden hamster eggs consists of recurring hyperpolarizations (Miyazaki & Igusa, 1981), in contrast to the potential in sea urchin or starfish eggs, which is a depolarization involving an overshoot and lasting for ∼10 min. The in vitro fertilization technique in mammals was developed during the 1970s, but no physiological recording of fertilization events had been performed. Each hyperpolarizing response (HR) in fertilized hamster eggs began from the resting potential of −25 mV and reached −65 mV, having a total duration of 10 s. A series of HRs occurred every 50–60 s and lasted for several hours. Since each HR was found to be due to a Ca2+-activated K+ conductance increase, HRs indirectly indicated transient repetitive increase in intracellular Ca2+ concentration ([Ca2+]i). HRs had been reported in fibroblasts and sympathetic ganglion neurons, and were a good indicator of Ca2+ signals at a time when direct measurement of [Ca2+]i was unavailable except for giant cells such as medaka fish eggs. n nOur paper (Igusa & Miyazaki, 1983) suggested two significant areas for later study: for physiology, the linkage of Ca2+ influx from outside of the cell to intracellular Ca2+ release from Ca2+ stores for the generation of long-lasting repetitive [Ca2+]i increase, which was later called intracellular Ca2+ oscillation, and for biology, Ca2+ oscillations that turned out to be the egg-activating signal characteristic of the fertilization of mammalian eggs. The paper showed that intervals between HRs are shortened by increasing external [Ca2+] or by hyperpolarization produced by passing continuous current through an intracellular microelectrode, i.e. by increasing the chemical or electrical driving force for Ca2+ influx, respectively. Intervals are prolonged by lowering external [Ca2+] or by depolarization, and HRs cease in Ca2+-free medium. HRs produced by injection of Ca2+ into the egg suggested that Ca2+-induced Ca2+ release would occur from Ca2+ stores when [Ca2+]i reached a critical level. It was hypothesized that persistent Ca2+ entry plays a critical role in providing Ca2+ to refill Ca2+ stores and maintain a repetitive [Ca2+]i rise due to repeated Ca2+ release. n nIn the middle of the 1980s, [Ca2+]i measurement became possible in egg cells, which are larger than somatic cells. Repetitive [Ca2+]i rise was recorded in hamster eggs using a Ca2+-sensitive microelectrode (Igusa & Miyazaki, 1986), and a Ca2+ wave was exhibited in each Ca2+ transient by a Ca2+-imaging method with the Ca2+-binding luminescent protein aequorin previously injected into the egg and using a super-sensitive camera system (Miyazaki et al. 1986). Ca2+ imaging was much more advanced around 1990 by utilizing Ca2+-binding fluorescent dyes such as fura-2 and by utilizing computers, and became applicable to somatic cells. It became a general concept that Ca2+ waves serve as a spatial Ca2+ signal propagating through the cell and Ca2+ oscillations are a temporal Ca2+ signal providing frequency-encoded cell signalling. n nIn 1987–88, the ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP3R) were purified as Ca2+ release channels in the endoplasmic reticulum (ER). Ca2+ waves and Ca2+ oscillations in fertilized hamster eggs were shown to be due to Ca2+ release from the ER exclusively through IP3R, and based on Ca2+-induced Ca2+ release mediated by IP3R instead of RyR (Miyazaki et al. 1993). The idea proposed in 1983 was extended to a single IP3-sensitive Ca2+ pool/Ca2+ entry model (Berridge & Dupont, 1994) with theoretical simulation (De Young & Keizer, 1992) for Ca2+ oscillations in somatic cells. The idea was substantiated by the discovery of capacitative (or store-operated) Ca2+ influx in various cells: Ca2+ entry that is coupled with Ca2+ release (Parekh & Penner, 1997). The store-operated Ca2+ influx pathway has been extensively analysed to date. As to Ca2+ dynamics underlying HRs, later experiment gave evidence utilizing Mn2+ quenching of intracellular fura-2 in mouse eggs that Mn2+ added to the extracellular medium enters the cytoplasm through the store-operated Ca2+ influx pathway, and is sequestered into the ER, released from the ER through IP3R, and extruded to the exterior (Mohri et al. 2001). n nA dramatic increase of [Ca2+]i at fertilization has been recorded in a wide variety of eggs since the later half of the 1980s, and it is a pivotal signal for egg activation common to all species examined to date (see reviews by Stricker, 1999; Miyazaki, 2006). Unfertilized eggs are arrested at a certain stage of meiosis, and the [Ca2+]i increase at fertilization induces release from the arrest, i.e. egg activation. Ca2+ oscillations consisting of transient Ca2+ spikes turned out to be the common and characteristic Ca2+ response in mammalian eggs. It has been shown in mouse eggs that the initial several Ca2+ spikes cause release from the metaphase of the second meiosis by activation of Ca2+- and calmodulin-dependent kinase II (CaMK II) leading to inactivation of the metaphase promoting factor (MPF) (see review by Ducibella et al. 2006). In mammalian eggs, the male and female pronuclei are formed 5 h or longer after sperm–egg fusion. Later Ca2+ spikes are responsible for pronucleus formation via reduction of mitogen-activated protein kinase (MAPK) activity (Ducibella et al. 2006). A series of Ca2+ spikes cease at about the time of pronucleus formation, at the interphase of the cell cycle. Thus, the generation of Ca2+ oscillations is cell cycle dependent. n nIt is of prime importantance to identify the sperm factor that induces Ca2+ oscillation in the egg, as it is the egg-activating factor. Evidence shows that the Ca2+ oscillation-inducing protein (COIP) is driven from the sperm cytoplasm into the ooplasm upon sperm–egg fusion, instead of sperm–egg surface receptor interaction leading to Ca2+ release from the ER (see review by Swann et al. 2006). The current strong candidate of COIP is a novel isozyme ‘zeta’ of phspholipase C (PLCζ) that hydrolyses membrane phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. PLCζ is specifically expressed in the mammalian sperm, and possesses appropriate characteristics to be the sperm factor that is introduced into the egg, first triggers Ca2+ release, and maintains Ca2+ oscillations (Swann et al. 2006; Miyazaki, 2006). At present, the study of the egg-activating sperm factor is the most advanced in mammals, compared with other kinds of animals. It remains to be elucidated that PLCζ actually functions as the sole sperm-derived egg-activating factor in physiological fertilization. n nAfter our paper on repetitive HRs, unexpectedly extensive advance has come in the study of spatiotemporal Ca2+ dynamics involving Ca2+ release/Ca2+ influx coupling mechanism and Ca2+ oscillations as the key factor for egg activation and early embryonic development after fertilization of mammalian eggs.