The C. elegans eggshell.

In all animals, oocytes are surrounded by an extracellular matrix upon fertilization. This matrix serves similar purposes in each animal. It functions to mediate sperm binding, to prevent polyspermy, to control the chemical environment of the embryo, and to provide physical protection to the embryo as it developes. The synthesis of the C. elegans matrix, or eggshell, begins when the oocyte enters the spermatheca and is fertilized by a single sperm. The process of eggshell synthesis is thought to take place during the completion of the maternal meiotic divisions such that the multi-layered eggshell is completed by anaphase II. The synthesis of the eggshell occurs in a hierarchical pattern in which the outermost layers are synthesized first in order to capture and retain the innermost layers as they form. Recent studies have revealed that the lipid-rich permeability barrier is distinct from the outer trilaminar eggshell. These new findings alter our previous understanding of the eggshell. This chapter aims to define each of the eggshell layers and the molecules that are known to play significant roles in their formation.

Functional redundancy of paralogs of an anaphase promoting complex/cyclosome subunit in Caenorhabditis elegans meiosis.

The anaphase promoting complex/cyclosome (APC/C) mediates the metaphase-to-anaphase transition by instructing the ubiquitination and turnover of key proteins at this stage of the cell cycle. We have recovered a gain-of-function allele in an APC5 subunit of the anaphase promoting complex/cyclosome. This finding led us to investigate further the role of APC5 in Caenorhabditis elegans, which contains two APC5 paralogs. We have shown that these two paralogs, such-1 and gfi-3, are coexpressed in the germline but have nonoverlapping expression patterns in other tissues. Depletion of such-1 or gfi-3 alone does not have a notable effect on the meiotic divisions; however, codepletion of these two factors results in meiotic arrest. In sum, the two C. elegans APC5 paralogs have a redundant function during the meiotic divisions.

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.

Components of the spindle assembly checkpoint regulate the anaphase-promoting complex during meiosis in Caenorhabditis elegans.

Temperature-sensitive mutations in subunits of the Caenorhabditis elegans anaphase-promoting complex (APC) arrest at metaphase of meiosis I at the restrictive temperature. Embryos depleted of the APC co-activator FZY-1 by RNAi also arrest at this stage. To identify regulators and potential substrates of the APC, we performed a genetic suppressor screen with a weak allele of the APC subunit MAT-3/CDC23/APC8, whose defects are specific to meiosis. Twenty-seven suppressors that resulted in embryonic viability and larval development at the restrictive temperature were isolated. We have identified the molecular lesions in 18 of these suppressors, which correspond to five genes. In addition to a single intragenic suppressor, we found mutations in the APC co-activator fzy-1 and in three spindle assembly checkpoint genes, mdf-1, mdf-2, and mdf-3/san-1, orthologs of Mad1, Mad2, and Mad3, respectively. Reduction-of-function alleles of mdf-2 and mdf-3 suppress APC mutants and exhibit pleiotropic phenotypes in an otherwise wild-type background. Analysis of a single separation-of-function allele of mdf-1 suggests that MDF-1 has a dual role during development. These studies provide evidence that components of the spindle assembly checkpoint may regulate the metaphase-to-anaphase transition in the absence of spindle damage during C. elegans meiosis.

Proteomic analysis of sperm regions that mediate sperm-egg interactions.

The sperm interacts with three oocyte-associated structures during fertilization: the cumulus cell layer surrounding the oocyte, the egg extracellular matrix (the zona pellucida), and the oocyte plasma membrane. Each of these interactions is mediated by the sperm head, probably through proteins both on the sperm surface and within the acrosome, a specialized secretory granule. In this study, we have used subcellular fractionation in order to generate a proteome of the sperm head subcellular compartments that interact with oocytes. Of the proteins we identified for which a gene knockout has been tested, a third have been shown to be essential for efficient reproduction in vivo. Many of the other presently untested proteins are likely to have a similarly important role. Twenty-five percent of the cell surface fraction proteins are previously uncharacterized. We have shown that at least two of these novel proteins are localized to the sperm head. In summary, we have identified over 100 proteins that are expressed on mature sperm at the site of sperm-oocyte interactions.

Defects in secretory pathway trafficking during sperm development in Adam2 knockout mice.

Adam2-null and Adam3-null male mice exhibit reduced levels of one or more ADAM proteins on mature sperm, in addition to the loss of the genetically targeted protein. ADAM protein loss was believed to occur posttranslationally, although the timing of loss and the mechanism by which the loss occurred were not explored. In this study we have found that in Adam3-null mice, fertilin beta (also known as ADAM2) is lost during the formation of testicular sperm. In Adam2-null males, most cyritestin (ADAM3) protein is also lost at this stage, but 25% of cyritestin is lost later, during sperm passage through the epididymis. Although normal levels of cyritestin are synthesized and acquire Endoglycosidase H resistance, indicating transit through the Golgi, the protein does not reach the cell surface. We also discovered that the majority of both fertilin beta and cyritestin are found in a Triton X-100 insoluble compartment on testicular sperm, when most of the cyritestin was observed on the cell surface. This insoluble compartment may represent a sorting platform, because in Adam2-knockout cells, only a small fraction of the cyritestin becomes Triton X-100 insoluble. Thus, it appears that cyritestin loss in Adam2-knockout mice may result, at least in part, from a disruption in protein trafficking.

Sperm-egg fusion: events at the plasma membrane.

Sperm-egg fusion is a cell-cell membrane fusion event essential for the propagation of sexually reproducing organisms. In gamete fusion, as in other fusion events, such as virus-cell and intracellular vesicle fusion, membrane fusion is a two-step process. Attachment of two membranes through cell-surface molecules is followed by the physical merger of the plasma membrane lipids. Recent progress has demonstrated an essential role for an oocyte tetraspanin, CD9, in mouse sperm-egg fusion, and a specific molecular site crucial for CD9 function has been identified. Absence of glycosylphosphatidylinositol-anchored proteins on the oocyte surface also results in loss of oocyte fusion competence in this gamete. These discoveries provide a strong starting point for the identification of additional proteins that have roles in sperm-egg fusion.