Stephen A. Stricker

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest—particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Application of Electrochemical Biosensors for Detection of Food Pathogenic Bacteria

Current practices for preventing microbial diseases rely upon careful control of various kinds of pathogenic bacteria in food safety and environmental monitoring. The main disadvantages of conventional bacterial detection methods are the multistep procedure and long time requirements. This article gives an overview of alternative electrochemical biosensors for detection of pathogenic bacteria in the food industry. Focus has been on new microbial metabolism-based, antibody-based and DNA-based biosensors. The underlying principles and applications of these biosensors are discussed. Recent developments in flow-injection biosensor systems with an electrochemical detection are also presented.

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs☆

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

Introduction to confocal microscopy and three-dimensional reconstruction.

Publisher Summary This chapter introduces the principle of confocal microscopy. The different types of confocal microscopes currently available and the various applications of confocal microscopy are discussed. Methods of specimen preparation for confocal microscopy are provided as guidelines and should be generally applicable to most cell types. The chapter also describes three-dimensional reconstruction, four-dimensional imaging, and the methodology for producing color prints and slides of confocal data. In conventional microscopy, much of the depth or volume of the specimen is uniformly and simultaneously illuminated in addition to the plane in which the objective lens is focused. This leads to out-of-focus blur from areas above and below the focal plane of interest. Out-of-focus light reduces contrast and decreases resolution, making it difficult to discern various cellular structures. In contrast, the illumination in a confocal microscope is not simultaneous, but sequential. The illumination is focused as a spot on one volume element of the specimen at a time.

Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF.

Before a proper Ca2+ response is produced at fertilization, oocytes typically undergo a maturation process during which their endoplasmic reticulum (ER) is restructured. In marine protostome worms belonging to the phylum Nemertea, the ER of maturing oocytes forms numerous distinct clusters that are about 5 μm in diameter. After fertilization, mature oocytes with such aggregates generate a normal series of Ca2+ oscillations and eventually disassemble their ER clusters at around the time that the oscillations cease. Immature oocytes, however, lack prominent ER clusters and fail to exhibit repetitive Ca2+ oscillations upon insemination, collectively suggesting that cell cycle-related changes in ER structure may play a role in Ca2+ signaling. To assess the effects of meiotic regulators on the morphology of the ER and the type of Ca2+ response that is produced at fertilization, nemertean oocytes were treated with pharmacological modulators of mitogen-activated protein kinases (MAPKs) or maturation-promoting factor (MPF) prior to confocal microscopic analyses. Based on such imaging studies and correlative assays of kinase activities, MAPKs of the ERK1/2 type (extracellular signal regulated kinases 1/2) do not seem to be essential for either structural reorganizations of the ER or repetitive Ca2+ signaling at fertilization. Conversely, MPF levels appear to modulate both ER structure and the capacity to produce normal Ca2+ oscillations. The significance of these findings is discussed with respect to other reports on ER structure, MPF cycling and Ca2+ signaling in oocytes of deuterostome animals.

Confocal laser scanning microscopy of calcium dynamics in living cells

Confocal laser scanning microscopy (CLSM) is widely used to monitor intracellular calcium levels in living cells loaded with calcium‐sensitive fluorophores. This review examines the basic advantages and limitations of CLSM in in vivo imaging analyses of calcium dynamics. The benefits of utilizing ratioed images and dextran‐conjugated fluorophores are addressed, and practical aspects of handling confocal datasets are outlined. After considering some relatively new microscopical methods that can be used in conjunction with conventional CLSM, possible future applications of confocal techniques in analyses of intracellular calcium dynamics are discussed. Microsc. Res. Tech. 46:356–369, 1999.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca2+) in all animals that have been examined, and such Ca2+ elevations, in turn, provide key activating signals that are required for non‐parthenogenetic development. Several lines of evidence indicate that the Ca2+ transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad‐based analysis of Ca2+ dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non‐mammalian species, and subsequently reviews various topics related to a sperm‐specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca2+ signals in fertilized oocytes or eggs are compared in a taxon‐by‐taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization‐induced Ca2+ signals are typically propagated as global waves that depend on Ca2+ release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5‐trisphosphate (IP3). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less‐common modes of producing Ca2+ transients are also examined. In addition, the importance of fertilization‐induced Ca2+ signals for activating development is underscored by noting some major downstream effects of these signals in various animals. Mol. Reprod. Dev. 80: 787–815, 2013.

Multiple triggers of oocyte maturation in nemertean worms: The roles of calcium and serotonin

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

STYLET FORMATION IN NEMERTEANS

Stylet formation was examined in nine species of nemerteans by light micros copy. The first stylets produced by larvae are assembled intracellularly over a period of several days within styletocytes of reserve stylet sacs. A reserve stylet is moved to the lumen of the proboscis, apparently by muscular contractions, and placed on the basis to become the central stylet. The stylets of adult nemerteans are also formed in styletocytes of reserve stylet sacs, and, depending on the species, reach full length in two to eight weeks. At the onset of styletogenesis, a membrane-bound vacuole develops in the styletocyte, and an organic matrix is formed at one edge of the vacuole. The calcified cortex of the stylet shaft is subsequently deposited around the organic matrix, and a knob-shaped proximal piece is formed on the shaft. Most adult nemerteans contain at least one developing stylet, and the rate of stylet formation is about the same in starved worms as it is in worms that have recently captured prey. Replacement of the central stylet occurs following prey attack, and occasionally when the worm is not feeding.

Stimulators of AMP-Activated Kinase (AMPK) Inhibit Seawater- But Not cAMP-Induced Oocyte Maturation in a Marine Worm: Implications for Interactions Between cAMP and AMPK Signaling

Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (=“germinal vesicle breakdown”, GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP‐activated kinase (AMPK), a well‐documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up‐ or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW‐ or cAMP‐induced GVBD, given that the catalytic α‐subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK‐mediated phosphorylation of acetyl‐CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice‐cold calcium‐free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW‐ or SW‐solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD‐regulating mechanism involving AMPK deactivation by cAMP‐mediated S485/491 phosphorylation. Mol. Reprod. Dev. 77: 497–510, 2010.