Chris Patton

A Practical Guide to the Preparation of Ca2+ Buffers

Calcium (Ca(2+)) is a critical regulator of an immense array of biological processes, and the intracellular [Ca(2+)] that regulates these processes is ~ 10,000 lower than the extracellular [Ca(2+)]. To study and understand these myriad Ca(2+)-dependent functions requires control and measurement of [Ca(2+)] in the nano- to micromolar range (where contaminating Ca(2+) is a significant problem). As with pH, it is often essential to use Ca(2+) buffers to control free [Ca(2+)] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca(2+) buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca(2+) buffers. These include pH dependence, selectivity for Ca(2+) (e.g., vs. Mg(2+)), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca(2+) buffering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca(2+)-buffered solutions for a wide array of biological applications.

Chapter 1 - A Practical Guide to the Preparation of Ca2 + Buffers

Calcium (Ca2+) is a critical regulator of an immense array of biological processes, and the intracellular [Ca2+] that regulates these processes is ~ 10,000 lower than the extracellular [Ca2+]. To study and understand these myriad Ca2+-dependent functions requires control and measurement of [Ca2+] in the nano- to micromolar range (where contaminating Ca2+ is a significant problem). As with pH, it is often essential to use Ca2+ buffers to control free [Ca2+] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca2+ buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca2+ buffers. These include pH dependence, selectivity for Ca2+ (e.g., vs. Mg2+), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca2+ buffering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca2+-buffered solutions for a wide array of biological applications.

Calmodulin activates NAD kinase of sea urchin eggs: An early event of fertilization

NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca2+ and calmodulin in vitro. The evidence is the requirement for low amounts of Ca2+ (Kd for Ca2+ of 4 x 10(-7) M) and the dissociation of a heat-stable activator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectrophoresis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperazine, an inhibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artificial parthenogenesis regimes that increase cytosolic Ca2+, but not by ammonia activation which only partially activates eggs and bypasses the Ca2+-rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca2+ at fertilization and the turning on of egg metabolism.

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.

Is there a role for the Ca2+ influx during fertilization of the sea urchin egg?

Abstract Both isotopic and microelectrode studies reveal a significant Ca2+ influx at fertilization which if freely distributed in the cytoplasm would equal 1–2 × 10−5 M. The role, if any, of this influx is disputed. We have attempted to reevaluate contradictory findings by others on this role. Our results with Strongylocentrotus purpuratus and Lytechinus pictus eggs, assessing fertilization with acrosome-reacted sperm in EGTA-buffered media (free [Ca2+], 4.4 × 10−8 M) indicate that exogenous Ca2+ is not required for fertilization and subsequent cleavage. The contradictory findings by others may have resulted from reduced fertilizability in Ca2+-free seawater, which can be circumvented by higher sperm concentration and by a sensitivity to temperature in Ca2+-free medium, which can be bypassed by carrying out fertilization at lower temperature. Also consistent with the absence of a requirement for this Ca2+ influx, we found that Ca2+ uptake can be induced in eggs by depolarizing the membrane with high [K+], but there is no resultant activation of egg metabolism. Under our conditions for fertilization in Ca2+-free media, there is no effect on the block to polyspermy but the initiation of the cortical reaction may be delayed. The data support the hypothesis that sperm induce release of Ca2+ from intracellular stores, perhaps by affecting an equilibrium between Ca2+ sequestration and Ca2+ release.

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata, Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.

Bacterial Symbionts Colonize the Accessory Nidamental Gland of the Squid Loligo opalescens via Horizontal Transmission

The accessory nidamental gland (AN gland), a reproductive organ of the mature female squid Loligo opalescens, harbors a dense culture of bacteria of unknown function. A multilayered sheath surrounding the L. opalescens egg case is similarly colonized by bacteria that presumably originate in the AN gland, as evidenced by their presence in the egg case at oviposition. This study investigates how these bacteria are transmitted to juvenile squid and examines some morphological consequences of bacterial colonization of AN gland tissues. By observing the structure of the AN gland in adults and the development and bacterial colonization of the gland in juveniles raised in captivity, we determined that the AN gland was absent in newly hatched squid and did not appear until 87 days post-hatching. At 129 days posthatching, the organ displayed tubules composed of a single layer of epithelial cells and expressing numerous cilia and microvilli. These tubules were not yet fully formed and thus were open to the mantle cavity and external seawater, possibly to aid in the acquisition of microorganisms. Since the AN gland developed a considerable time after hatching, it most likely acquires its symbionts horizontally from environmental seawater and not vertically from the egg case sheath. The switch from expression of cilia to production of microvilli on the epithelial cell surface may dictate the competence of the tissue for bacterial colonization. Electron microscopic examination of juvenile and adult AN glands revealed that an analogous process occurs during the development of the related light organ of other cephalopod species that harbor symbiotic bacteria.

A morphological study of nonrandom senescence in a colonial urochordate.

Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.

Phosphoinositide metabolism at fertilization of sea urchin eggs measured with a GFP‐probe

Fertilization elicits a dramatic, transient rise in Ca2+ within the egg which is an essential component of egg activation and consequent initiation of development. In the sea urchin egg, three distinct Ca2+ stores have been identified which could, either individually or in combination, initiate Ca2+ release at fertilization. Inositol 1,4,5‐trisphosphate (IP3) production by phospholipase C (PLC) has been suggested as the singular signal in initiating the Ca2+ transient. Other studies indicate that Ca2+ stores gated by cyclic adenosine diphosphate ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP) are also necessary. We have examined the temporal relationship between the Ca2+ rise and IP3 production at fertilization in vivo within individual eggs using a green fluorescent protein (GFP) coupled to a pleckstrin homology (PH) domain that can detect changes in IP3. Translocation of the probe occurred after the Ca2+ rise was initiated. Earlier, and possibly smaller, IP3 changes could not be excluded due to limitations in probe sensitivity. High IP3 levels are maintained during the decline in cytoplasmic Ca2+, suggesting that later IP3 metabolism might not be related to regulation of Ca2+, but may function to modulate other PIP2 regulated events such as actin polymerization or reflect other novel phosphoinositide signaling pathways.

Cortical Granules of Sea Urchin Eggs do not Undergo Exocytosis at the Site of Sperm‐egg Fusion*

Microscopic observations of sea urchin egg fertilization (phase contrast, Nomarski and transmission electron microscope) reveal that the cortical granules in the area of sperm egg‐fusion do not undergo exocytosis. These intact granules remain associated with the sperm, moving into the egg cytoplasm with the entering sperm. This sperm‐cortical granule association occurs before the sperm centriole affects microtubule organization and the sperm‐cortical granule association is not affected by cytochalasin D or griseofulvin. We discuss the possibility that a reorganization of the egg cytoplasm ensues from the sperm‐egg interaction at the site of sperm‐egg fusion. Other possibilities are that the retention of cortical granules is not related to egg reorganization, but is necessary for successful sperm incorporation or reflects an unrelated component of the activation process.