Tomohide Shikano

The gamete fusion process is defective in eggs of Cd9-deficient mice.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins, and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9−/− mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9−/− females. Intracellular Ca2+ oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

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.

Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development.

Direct injection of a single spermatozoon into an oocyte (ICSI) can produce apparently normal offspring. Although the production of normal offspring by ICSI has been successful in mice and humans, it has been less successful in many other species. The reason for this is not clear, but could be, in part, due to inconsistent activation of oocytes because of delayed disintegration of sperm plasma membrane within oocytes and incorporation of the acrosome containing a spectrum of hydrolyzing enzymes. In the mouse, the removal of sperm plasma membrane and acrosome was not a prerequisite to produce offspring by ICSI, but it resulted in earlier onset of oocyte activation and better embryonic development. The best result was obtained when spermatozoa were demembranated individually immediately before ICSI by using lysolecithin, a hydrolysis product of membrane phospholipids.

Difference in Ca2+ Oscillation-Inducing Activity and Nuclear Translocation Ability of PLCZ1, an Egg-Activating Sperm Factor Candidate, Between Mouse, Rat, Human, and Medaka Fish

Mouse phospholipase C, zeta 1 (PLCZ1), a strong candidate of egg-activating sperm factor, induces Ca(2+) oscillations and accumulates into formed pronucleus (PN) when expressed by cRNA injection. These activities were compared among mouse and human PLCZ1, newly cloned rat Plcz1, and medaka fish plcz1. The PLCZ1 proteins of the four species have an approximately homologous sequence of nuclear localization signal. However, the nuclear translocation ability was defective in rat, human, and medaka PLCZ1 expressed in mouse eggs. Rat PLCZ1 could not enter rat PN, whereas mouse PLCZ1 could. Mouse and human PLCZ1 translocated into the nucleus of COS-7 cells transfected with cDNA. There was little medaka PLCZ1 accumulated in the nucleus, and rat PLCZ1 was never located in the nucleus. All PLCZ1 proteins including fish could induce Ca(2+) oscillations in mouse eggs, but the activity was variable in the order of human >> mouse > medaka >> rat, estimated from minimal RNA concentration to induce Ca(2+) spikes. Ca(2+) oscillations by human PLCZ1 continued far beyond the time of PN formation (T(PN)), whereas those by mouse PLCZ1 ceased slightly before T(PN). High-frequency Ca(2+) spikes by overexpressed rat PLCZ1 stopped far before T(PN), possibly by feedback inhibition. Ca(2+) oscillations by fertilization of rat eggs stopped at T(PN), despite defective nuclear translocation of rat PLCZ1. Thus, PLCZ1 sequestration into PN participates in termination of Ca(2+) oscillations at the interphase of mouse embryos but does not always operate in other mammals, notably in rat embryos.

The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor

Sperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, or Trp13, Phe14, or Val18 in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Activity of a Sperm-Borne Oocyte-Activating Factor in Spermatozoa and Spermatogenic Cells from Cynomolgus Monkeys and Its Localization after Oocyte Activation

Abstract It is widely accepted that mature mammalian oocytes are induced to resume meiosis by a sperm-borne oocyte-activating factor(s) (sperm factor, SF) immediately after normal fertilization or intracytoplasmic sperm injection. The SF is most likely a soluble factor that is localized within the cytoplasm of mature spermatozoa, but the exact stage at which it appears during spermatogenesis and its localization after oocyte activation is not fully understood, except in the mouse. First, we injected mature spermatozoa and spermatogenic cells from cynomolgus monkeys into mouse oocytes to assess their oocyte-activating capacity. More than 90% of mouse oocytes were activated after injection of monkey spermatozoa. Round spermatids and primary spermatocytes (late pachytene to diplotene) also activated oocytes (93% and 79%, respectively). Injection of monkey spermatozoa and spermatids induces intracellular Ca2+ oscillations in a pattern similar to that seen following normal fertilization. Most spermatocytes did not produce typical intracellular Ca2+ oscillations. Second, we transferred pronuclei or cytoplasts from mouse oocytes that had been activated by monkey spermatozoa or spermatids into intact mature mouse oocytes by electrofusion in order to examine the localization of the SF after pronuclear formation. Some of the SF was localized within the pronuclei, but some stayed in the ooplasm. This study demonstrated that spermatogenic cells of cynomolgus monkeys acquire oocyte-activating capacity at much earlier stages than those of mice, and that the monkey SF has a pronucleus-directing nature, although to a lesser extent than the mouse SF.

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.