Identification and role of carbohydrates on the surface of gametes in the zebra mussel, Dreissena polymorpha.

The objective of this study is to identify surface carbohydrates on zebra mussel, Dreissena polymorpha, eggs and sperm and to analyze their potential role in fertilization. The lectins WGA, Con A, LcH, LTA, SBA, PNA, and GSII were tested for affinity to both eggs and sperm. WGA, Con A, and LcH uniformly labeled eggs. LTA, SBA, PNA, and GSII did not. WGA labeled the entire sperm surface including the unreacted acrosome. Labeling by Con A, LcH, LTA, SBA, PNA, and GSII was restricted to the inner acrosomal region of acrosome-reacted sperm. GSII labeling suggests the presence of N-acetyl-d-glucosamine (GlcNAc) only in the inner acrosomal membrane and not on eggs. GlcNAc blocked sperm-egg binding. GSII labeling was associated with a ring-like structure at the site of sperm entry intimately associated with sperm-egg binding. Nonfertilizing sperm were detached from the egg surface along with the GSII basal ring about 15 min postinsemination in a process blocked by trypsin inhibitors.

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas.

The plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

Sperm incorporation and pronuclear development during fertilization in the freshwater bivalve Dreissena polymorpha.

The invasive zebra mussel, Dreissena polymorpha (D. polymorpha), is proving to be a valuable model for understanding general mechanisms of fertilization, particularly regarding sperm incorporation. In the present study, we tracked the various components of the fertilizing sperm of D. polymorpha during sperm incorporation. During fertilization the sperm membrane remains associated with the egg surface as a distinct patch that disperses over time. This patch marked the site of sperm entry that occurs predominately on the CD blastomere. Taking advantage of the relatively unpigmented cytoplasm, real-time observations were made of the incorporated sperm nucleus as it decondensed and reformed as a developing pronucleus. Pronuclear enlargement occurred progressively and at rates comparable with previously reported fixed-time point observations. Sperm mitochondria were incorporated and separated from the sperm along the leading edge of the decondensing nucleus. Sperm mitochondria labeled with Mitotracker Green remained predominately associated with the CD blastomere following first cleavage and could be tracked to the 16-cell stage before the fluorescence was too faint to detect. Additionally, the demembranated sperm axoneme was incorporated, separated during nuclear decondensation, and remained visible in the egg cytoplasm up to 30 min postinsemination (PI). The present study provides one of the most complete descriptions of incorporation on multiple sperm components into the egg and coordinates fixed-time point observations with real-time observations of sperm within the remarkably transparent egg cytoplasm of zebra mussels.

Protein transport and signal transduction during fertilization in chlamydomonas.

Fertilization in Chlamydomonas begins with flagellar adhesion between mating type plus and mating type minus gametes and is consummated within minutes by zygote formation. Once fusion occurs, the newly merged gametes cease existence as distinct entities, and the diploid zygote immediately initiates transcription of zygote-specific genes. Accomplishing fertilization within such a short time requires the rapid and signaled movement of pre-existing membrane and cytoplasmic proteins between and within several cellular compartments. Generation within the adhering flagella of the initial signals for protein movement, as well as movement itself of at least one cytoplasmic protein from the cell body to the flagella, depend on the microtubule motor, kinesin-II and presumably on intraflagellar transport (IFT). Adhesion and fusion of the two gametes depend on a second translocation event, the movement of an adhesion/fusion protein onto the surface of a rapidly elongating, microvillous-like fusion organelle. Finally, the merging of the two separate gametes, each containing sex-specific proteins, into a single cell allows the formerly separate proteins to form new interactions that regulate zygote development. Two proteins - a nuclease and a homeodomain protein - which were present only in the plus gamete, are 'delivered' to the cytoplasm of the zygote during gamete fusion. The nuclease is selectively imported into the minus chloroplast, where it degrades the chloroplast DNA, thereby ensuring uniparental inheritance of plus chloroplast traits. The homeodomain protein binds with an as yet unidentified protein delivered by the minus gamete, and the new complex activates transcription of zygote-specific genes.

The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes.

The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mt+ and mt- gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a approximately 95-kDa protein present on the external surface of both unactivated and activated mt+ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mt+ gametes at the site of cell-cell fusion and essential for an early step in the fusion process.