Structural analysis of fertilization in the fish Brycon orbignyanus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sporophytic apomixis in polyploid Anemopaegma species (Bignoniaceae) from central Brazil

Apomixis and polyploidy have been important in the evolution of the angiosperms, and sporophytic apomixis has been associated with polyembryony and polyploidy in tropical floras. We studied the occurrence of polyembryony in populations of tetraploid Anemopaegma acutifolium, A.arvense and A.glaucum from the Brazilian cerrados, and histological features of sexual and apomictic processes were investigated in A.acutifolium. All populations and species were polyembryonic (68.9-98.4% of seeds). Normal double fertilization occurred in most ovules, with exceptions being that 3% of ovules were penetrated but not fertilized and in 4% of ovules both synergids were penetrated. The penetration of both synergids suggests a continuous attraction of pollen tubes and polyspermy. Adventitious embryo precursor cells (AEPs) arose from nucellar and integumental cells of the ovule in pollinated and unpollinated A.acutifolium, indicating sporophytic apomixis. However, further embryo and endosperm development required pollination and fertilization. This pseudogamy also allows concurrent sexual embryo development. Similar polyembryony rates and polyploidy indicated that A.arvense and A.glaucum are also apomictic, forming an agamic complex similar to that observed for some species of confamilial, but not closely related Handroanthus. The co-occurrence of apomixis and polyploidy in different groups of Bignoniaceae indicates homoplasious origin of these agamic complexes. © 2013 The Linnean Society of London.

Expression of α6 integrin subunit in bovine oocyte and its potential role during fertilization

Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.

Funktionelle und strukturelle Charakterisierung des Ovastacins in vivo und in vitro

Die Metalloprotease Ovastacin, ein Vertreter der Astacin-Familie, wurde erstmals 2004 beschrieben. Im Ovar von Säugetieren ist Ovastacin-mRNA im Zeitfenster vom Stadium der Sekundärfollikel bis kurz nach der Befruchtung der Eizelle zu finden. Der Expressionsort und -zeitpunkt sowie die Sequenzähnlichkeit von über 60% mit sogenannten „Schlüpfenzymen“ (engl. hatching enzymes), die man in den Eizellen und Zygoten niederer Wirbeltiere und Wirbelloser gefunden hatte, ließen die Vermutung aufkommen, es könnte sich hier um das Säugerhomolog dieser Proteasen handeln. Generell lösen hatching Enzyme die derben embryonalen Hüllstrukturen (bei Säugern die Zona pellucida, ZP) beim Schlüpfvorgang auf. Die essentielle Bedeutung des Ovastacins für die Befruchtung wird durch die um ca. 30% reduzierte Fruchtbarkeit von Ovastacin defizienten Mäusen belegt. Hochinteressant war in diesem Zusammenhang die Entdeckung des Ovastacins in den Cortikalgranula der Oocyten sowie seine Fähigkeit, das Zona pellucida Protein 2 zu schneiden. Die dadurch bewirkte Verhärtung der Zona pellucida verhindert das Eindringen weiterer Spermien, das heißt sie baut eine Barriere gegen Polyspermie auf. Ziel dieser Arbeit war es, Belege für die physiologische Funktion des Ovastacins zu finden. Vor allem galt es, potentielle Aktivatoren zu identifizieren, da das Enzym wie alle Astacine als inaktive Vorstufe gebildet wird, die proteolytisch aktiviert werden muss. Zu diesem Zweck exprimierte ich rekombinantes Pro-Ovastacin in Insektenzellen. Aktivierungsstudien in vitro zeigten, dass ein saures Milieu zu einer Aktivierung führt, ohne die Abspaltung des Propeptids zu bewirken. Sequenzalignments und ein homologes Strukturmodell des Ovastacins wiesen auf Trypsin- oder Elastase-ähnliche Serinproteasen als potentielle Aktivierungsenzyme hin. Tatsächlich konnte mit diesen beiden Proteasetypen zum ersten Mal aktives Ovastacin aus Pro-Ovastacin erzeugt werden. Trypsin kommt als physiologischer Aktivator allerdings nicht in Betracht, da es bisher in keinem der Gewebe nachgewiesen werden konnte, in dem Ovastacin exprimiert wird. Die neutrophile Elastase dagegen konnte in der Leber, im Herz sowie im Blutplasma nachgewiesen werden. Mit Hilfe spezifischer Antikörper konnte das Herz als Expressionsort für Ovastacin bestätigt werden. Somit wäre Elastase ein potentieller physiologischer Aktivator von Ovastacin. Die Identifikation des Ovastacins in Geweben wie Leber, Herz, Nabelschnur und im Blutplasma weist auf eine Rolle der Protease in proteolytischen Netzwerken außerhalb der Spermien-Ei-Interaktion hin. Die Bedeutung der biologischen Kontrolle des Ovastacins bei der Befruchtung der Säugereizelle wird durch die Beobachtung untermauert, dass das Leberprotein Fetuin B als physiologischer Ovastacininhibitor fungiert und dadurch eine vorzeitige Verhärtung der Zona pellucida verhindert, die andernfalls die Penetration von Spermien prinzipiell verhindern würde.

CO2-induced fertilization impairment in Strongylocentrotus droebachiensis collected in the Arctic

Fertilization depends on distribution and aggregation patterns of sea urchins which influence gamete contact time and may potentially enhance their vulnerability to ocean acidification. In this study, we conducted fertilization experiments to assess the effects of selected pH scenarios on fertilization success of Strongylocentrotus droebachiensis, from Spitsbergen, Arctic. Acidification was achieved by aerating seawater with different CO2 partial pressures to represent pre-industrial and present conditions (measured ~180-425 µatm) and future acidification scenarios (~550-800, ~1,300, ~2,000 µatm). Fertilization success was defined as the proportion of successful/unsuccessful fertilizations per treatment; eggs were classified according to features of their fertilization envelope (FE), hyaline layer (HL) and achievement of cellular division. The diagnostic findings of specific pathological aberrations were described in detail. We additionally measured intracellular pH changes in unfertilized eggs exposed for 1 h to selected acidification treatments using BCECF/AM. We conclude that (a) acidified conditions increase the proportion of eggs that failed fertilization, (b) acidification may increase the risk of polyspermy due to failures in the FE formation supported by the occasional observation of multiple sperms in the perivitelline space and (c) irregular formation of the embryo may arise due to impaired formation of the HL. The decrease in fertilization success could be also related to the observed changes in intracellular pH at pCO2 ~ 1,000 µatm or higher.

Reliably estimating the effect of toxicants on fertilization success in marine broadcast spawners

Recently, there has been a dramatic increase in the number of ecotoxicological studies examining the effects of toxicants on fertilization success in marine broadcast spawners and it appears that this life-history stage is one of the most vulnerable to toxicants. Most of the studies examining this issue use single sperm concentrations in their assays. Here, I discuss recent advances in fertilization ecology that suggest this technique has some severe limitations resulting in unreliable estimations of the size and direction of toxicant effects. I present an alternative assay technique and two metrics (F-max and [Sperm](max)) that will reliably estimate the size of a toxicant's effect on fertilization success. This technique has the added advantage of making comparisons among species and studies easier without an impractical increase in effort. (c) 2006 Elsevier Ltd. All rights reserved.