François Dubé

Activation of Barnea candida (mollusca, pelecypoda) oocytes by sperm or KCl, but not by NH4Cl, requires a calcium influx

Abstract Artificial activation of Barnea candida oocytes by NH 4 Cl and the addition of excess KCl are, respectively, independent and dependent upon external Ca 2+ concentration. The activating efficiency of NH 4 Cl increases when external pH is raised which suggests that the proportion of unionized penetrating NH 3 is the key factor for activation under these conditions. The external Ca 2+ -dependent period for KCl or sperm-induced activation does not exceed 3–4 min. The transmembrane Ca 2+ flux inhibitor D-600 reversibly inhibits KCl- or sperm-induced activation and the D-600-sensitive period closely corresponds to the Ca 2+ -dependent period. Ca 2+ ions alone can also trigger activation provided external Mg 2+ concentration is lowered. Finally, a direct demonstration is presented for the existence of a D-600-sensitive Ca 2+ uptake following KCl- or sperm-induced activation, by using the radioactive tracer 45 Ca. The significance of that Ca 2+ influx is discussed with respect to the possible involvement of an intracellular pH shift as a main factor in the activation of lamellibranch mollusc oocytes.

Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca, Pelecypoda) oocytes

Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KCl or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na+ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na+ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions.

Acid Release during Activation of Barnea candida (Mollusca, Pelecypoda) Oocytes

Barnea caridida oocytes release acid (1.35 pmole H+/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H+/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium‐acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium‐acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca++‐H+ exchange transport but appears more likely to be due to a Na+‐H* exchange as it has been demontrated in sea urchin eggs.

A new G protein-coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells

Biogenic amine receptors mediate wide‐ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic‐like receptors were previously characterized pharmacologically and found to engender control of the animals bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT‐PCR strategy, we obtained a full‐length cDNA encoding a polypeptide with typical G protein‐coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK‐ and COS‐7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11‐fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.

Mechanisms of calcium release and sequestration in eggs of Chaetopterus pergamentaceus

Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.

Localization and Quantification of Gonad Serotonin During Gametogenesis of the Surf Clam, Spisula solidissima

In the surf clam, Spisula solidissima, serotonin was reported to induce spawning when injected into the gonads. At nanomolar concentrations, it facilitates the fertilizability of oocyte by sperm, at micromolar concentration, it triggers the meiotic maturation of prophase 1-arrested oocytes, thus mimicking the effect of sperm. To further understand the role of serotonin in the gametogenic and spawning processes, we used both immunohistochemistry and high-pressure liquid chromatography linked with electrochemical detection to detect serotonin in the gonads of the surf clam. We found serotonin-containing varicose fibers covering the surface of the germinal epithelium in both sexes. The area occupied by the serotonergic innervation field encircling gonad acini varied according to the gonadal stages (active phase, ripe phase, partially spawned phase, spent phase). We also found large variations in the serotonin concentration between specimens during the gametogenic cycle. The serotonin concentration was correlated with gonad growth: it decreased in the ripe phase in comparison with the previous phase, the active phase. We attribute the decrease to the increase of total gonad mass in this stage. In contrast, as spawning begins, the total gonad mass declines while the gonad serotonin concentration increases to a level similar to that found in active phase. The finding that prior to spawning, serotonin is present in the gonads within fibers exhibiting distinct varicosities suggests that it is implicated in spawning.

Characterization of a novel octopamine receptor expressed in the surf clam Spisula solidissima.

We have cloned and sequenced a cDNA from the surf clam (Spisula solidissima, a pelecypod mollusc) that encodes an octopamine receptor which we have named Spi-OAR. The sequence of Spi-OAR shares many similarities with two Aplysia and three Drosophila octopamine receptors belonging to a sub-group of beta-adrenergic-like octopamine receptors. Using an expression vector and transient transfections of Spi-OAR into HEK 293 cells, we observed an increase of cAMP upon addition of octopamine and, to a lesser extent, of tyramine, but not after addition of dopamine, serotonin, or histamine. Using a battery of known agonists and antagonists for octopamine receptors, we observed a rather unique pharmacological profile for Spi-OAR through measurements of cAMP. Spi-OAR exhibited some constitutive activity in HEK 293 cells and no Ca(2+) responses could be detected following addition of octopamine to Spi-OAR-transfected cells. RT-PCR analysis revealed ubiquitous expression of Spi-OAR mRNA in all adult tissues, oocytes and early embryos examined. While addition of serotonin to isolated clam oocytes resulted in meiotic activation, similar additions of octopamine had no effect, suggesting that its potential role in clam reproductive physiology differs significantly from that of serotonin. This work identifies Spi-OAR as a novel mollusc octopamine receptor closely related to other invertebrate beta-adrenergic-like octopamine receptors, with possible reproductive and other physiological functions. This initial characterization of Spi-OAR makes possible further investigations and comparisons with more studied and familiar insect or gastropod mollusc octopamine receptors.

Ca2+ influx and stimulation of protein synthesis in sea urchin eggs.

Acid release, Ca2+ influx and stimulation of protein synthesis were investigated with sea urchin eggs submitted to an excess of KCl, to NH4Cl, and to a combination of both. KCl, though unable to promote any acid release, triggers a large 45Ca uptake by eggs and slightly stimulates protein synthesis, provided that external Ca2+ is present. NH4Cl, which induces an intracellular pH increase, triggers a late and small 45Ca uptake but highly stimulates protein synthesis. The combined use of NH4Cl + KCl allows a large 45Ca uptake to occur but the level of protein synthesis is similar to that obtained with NH4Cl alone and is identical whether external Ca2+ is present or not. In contrast to previous works, our results show that the large stimulation of protein synthesis triggered by an intracellular pH increase, as after NH4Cl activation, cannot be enhanced by a Ca2+ influx. This suggests that the Ca2+ influx occurring after fertilization has only a minimal effect on the overall stimulation of protein synthesis.

Trifluoperazine-sensitive step during sea urchin, echiuroid and pelecypod egg activation

Trifluoperazine, a calmodulin‐antagonist, is shown to inhibit egg activation by ionophore A 23187 in sea urchin (I50: 43 μM), by trypsin in echiufoids (I50: 22 μM) and by KCl in bivalves (I50: 34 μM). In each case the inhibition could be reversed by washing the eggs and the trifluoperazine‐sensitive period was clearly limited. In Barnea and Urechis, trifluoperazine inhibits calcium uptake. A common trifluoperazine‐sensitive step, possibly involving calmodulin, may thus be shared by a variety of animal groups during egg activation.

Cloning, sequencing, and expression analysis of mouse glucosamine-6-phosphate deaminase (GNPDA/oscillin).

It was reported that a hamster protein, called “oscillin,” with a sequence related to that of an Escherichia coli GNPDA triggered Ca2+ oscillations in mammalian oocytes when introduced into their cytoplasm upon fertilization. Recently, it was shown that GNPDA/oscillin is ubiquitously expressed in rat tissues and that a recombinant hamster GNPDA/oscillin protein does not exhibit oscillin activity when injected into oocytes. In the mouse, the nature and role of such a GNPDA/oscillin is not known, but another candidate protein, tr‐kit, has been proposed as a sperm factor causing oocyte activation. In order to clarify this issue, we have characterized the mouse homolog of hamster and human GNPDA/oscillin, and examined its expression along with that of tr‐kit, in parallel. We report here the molecular cloning and sequencing of mouse GNPDA/oscillin, which shows over 96% identity with the hamster and human homologs. Using specific primers, we performed an RT‐PCR analysis to determine the tissue distribution of mouse GNPDA/oscillin mRNA. Unlike tr‐kit mRNA which is expressed solely in mouse testis, GNPDA/oscillin mRNA is detected in unfertilized oocytes and in all tissues examined including testis, heart, thymus, liver, ovary, uterus, kidney, spleen, and lung. The protein itself is also detected in all tissues examined by Western blots. Indirect immunofluorescence studies, using an antibody raised against hamster GNPDA, demonstrate that GNPDA is lost with the acrosome reaction of mouse spermatozoa, is localized in the equatorial and neck regions of the human spermatozoa and the post‐acrosomal region of the hamster spermatozoa. Our results thus indicate that mouse GNPDA/oscillin, the homolog of hamster oscillin, unlike tr‐kit, does not exhibit some of the required characteristics expected from a putative sperm‐derived oocyte‐activating factor. Mol. Reprod. Dev. 56:424–435, 2000.