The double-edged sword of the mammalian oocyte--advantages, drawbacks and approaches for basic and clinical analysis at the single cell level.

Oocytes are usually the largest cells in the body and as such offer unique opportunities for single-cell analysis. Unfortunately, these cells are also some of the rarest in the mammalian female, usually necessitating single-cell analysis. In cases of infertility in humans, determining the quality of the oocyte is often restricted to a morphological analysis or to the study of cellular behaviors in the developing embryo. Minimally invasive approaches could greatly assist the clinician to prioritize oocytes for fertilization or following fertilization, which embryo to transfer back into the woman. Transcriptomics of human and mouse oocytes may have great utility, and recently it was learned that the polar body faithfully reflects the transcript prevalence in the oocyte. The polar body may thus serve as a minimally invasive proxy for an oocyte in the clinic. In the mouse, the transcriptomes of oocytes from mice of the same strain are markedly similar; no significant differences are apparent in transcript prevalence or identity. In human oocytes however, the transcript pool is highly variable. This is likely the result of different histories of each oocyte, in the age of the donor woman, the different hormonal exposures and the prolonged time from specification of the primary oocyte to the fully grown and ovulated egg. This variability in human oocytes also emphasizes the need for cell-by-cell analysis of the oocytes in vitro; which oocytes have a better potential for fertilization and development? To this end, new imaging capabilities are being employed. For example, a single-cell analytical device for oocytes (the simple perfusion apparatus, or SPA) enables investigators to load multiple oocytes in individual wells, to visualize them on the microscope and to use controlled temperature and media flow by perfusion for optimal clinical applications. Recently, developed Raman microspectroscopy approaches suggest that this imaging modality may enable more in-depth analysis of the molecular characteristics of an oocyte that, in combination with the SPA and transcriptomic approaches, might assist the clinician to prioritize more effectively human oocytes and embryos for transfer into women. This review is intended to update the reader on the status of the examination of single oocytes from a variety of approaches and to emphasize areas that may be primed for advancement in the near future.

Apoptosis in sea urchin oocytes, eggs, and early embryos.

Certain experimental manipulations with sea urchin oocytes, eggs, and early embryos result in induction of cell death. We were interested in whether or not these cells possessed functional apoptotic machinery, and whether cellular demise under certain experimental conditions is due to activation of a programmed cell death pathway. Therefore, we evaluated a number of apoptosis assays in sea urchin oocytes, eggs, and early embryos experimentally induced to apoptose with staurosporine. Our results indicate that these cells each possess and activate necessary apoptotic machinery that leads to characteristic apoptotic phenotypes. The eggs of this animal have completed meiosis, and are quiescent transcriptionally, translationally, and metabolically. Surprisingly, they still undergo apoptosis. The progression through apoptosis of treated specimens could be followed by morphological changes of the cells, by chromatin condensation and degradation, and by activation of caspases. The similarities and differences in the execution of apoptosis between the cell types studied are discussed. Results of this study will be useful for interpreting experiments in these model systems in which different molecules are targeted for interference and which brings about cell death.

The biology of cortical granules.

An egg-that took weeks to months to make in the adult-can be extraordinarily transformed within minutes during its fertilization. This review will focus on the molecular biology of the specialized secretory vesicles of fertilization, the cortical granules. We will discuss their role in the fertilization process, their contents, how they are made, and the molecular mechanisms that regulate their secretion at fertilization. This population of secretory vesicles has inherent interest for our understanding of the fertilization process. In addition, they have import because they enhance our understanding of the basic processes of secretory vesicle construction and regulation, since oocytes across species utilize this vesicle type. Here, we examine diverse animals in a comparative approach to help us understand how these vesicles function throughout phylogeny and to establish conserved themes of function.

Membrane trafficking machinery components associated with the mammalian acrosome during spermiogenesis.

Active trafficking from the Golgi apparatus is involved in acrosome formation, both by delivering acrosomal contents to the nascent secretory vesicle and by controlling organelle growth and shaping. During murine spermiogenesis, Golgi antigens (giantin, beta-COP, golgin 97, mannosidase II) are detected in the acrosome until the late cap-phase spermatids, but are not found in testicular spermatozoa (maturation-phase spermatids). This suggests that Golgi-acrosome flow may be relatively unselective, with Golgi residents retrieved before spermiation is complete. Treatment of spermatogenic cells with brefeldin A, a drug that causes the Golgi apparatus to collapse into the endoplasmic reticulum, disrupted the Golgi in both pachytene spermatocytes and round spermatids. However, this treatment did not affect the acrosomal granule, and some beta-COP labeling on the acrosome of elongating spermatids was maintained. Additionally, N-ethylmaleimide sensitive factor, soluble NSF attachment proteins, and homologues of the t-SNARE syntaxin and of the v-SNARE VAMP/synaptobrevin, as well as members of the rab family of small GTPases, are associated with the acrosome (but not the acrosomal granule) in round and elongated spermatids. This suggests that rab proteins and the SNARE machinery for membrane recognition/docking/fusion may be involved in trafficking during mammalian acrosome biogenesis.

Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea Urchin.

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.

Syntaxin, VAMP, and Rab3 are selectively expressed during sea urchin embryogenesis.

SNARE and rab protein family members were originally identified in terminally differentiated cell types. These proteins are phylogenetically conserved and while compelling evidence demonstrates their involvement in the secretory pathway, their exact function is debated. We recently identified SNARE protein family members in the sea urchin egg and provided evidence that rab3 functions in the exocytosis of cortical granules. Here we tested the hypothesis that these same proteins might also be present throughout embryogenesis to mediate membrane fusion events. We provide evidence that the sea urchin possesses a low complexity of gene family members of syntaxin, VAMP, and rab3 and that these proteins are not only present during development, but are enriched in regions of the embryo with active secretory roles. We found accumulation of each family member in the apical and basal aspects of cleaving blastomeres, indicative of bidirectional secretion into the extraembryonic environment and blastocoel. Elevated levels of syntaxin, VAMP, and rab3 were also found in the mesodermally derived pigment cells that invade and move within the ectoderm. These cells likely rely on SNARE and rab proteins to enable mobility by mediating the secretion of enzymes that break adhesion to neighboring cells and the extracellular matrix. In addition, these secretory proteins are enriched in the gut following gastrulation. Thus, we conclude that VAMP, syntaxin, and rab3 mediate a variety of secretory events that is important for development.

ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

SFE1, a constituent of the fertilization envelope in the sea urchin is made by oocytes and contains low-density lipoprotein-receptor-like repeats.

At fertilization in most animals, cortical granules of the egg or oocyte secrete their contents, whose function it is to modify the extracellular matrix. This modified matrix then participates in the block to polyspermy and protection for early embryonic development. In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination. Several of these content proteins then bind to the nascent vitelline layer of the egg and lift off the cell surface to form a stable, impervious, fertilization envelope. At least six major proteins are present in the envelope, and recently we have identified cDNA clones of two, ovoperoxidase, and SFE9. Here we report on the identification and characterization of SFE1, a constituent of the fertilization envelope of the sea urchin Strongylocentrotus purpuratus, that has revealing characteristics of how the envelope might form and what protein interaction domains might predominate. We present the largest cDNA sequence we were able to identify representing approximately two thirds of the predicted protein coding region. The C-terminal half of the cognate SFE1 protein contains two different amino acid repeat motifs: a cysteine-rich (15%) motif of 40 amino acids that is tandemly repeated 22 times and is followed by a serine/threonine-rich (38%) repeat of 63 amino acids that is tandemly repeated 3.5 times. Surprisingly, just N-terminal to the cysteine-rich repeat region is a sequence of five repeats with similarity to repeats in another cortical granule protein, SFE9, and to the motif originally identified in the receptor of low-density lipoproteins, the LDLr motif. The amino acid composition deduced from the partial SFE1 cDNA is similar also to the composition of proteoliaisin, a protein thought to tether the ovoperoxidase to the vitelline layer of the egg and thereby sequester the crosslinking activity of the ovoperoxidase to a limited population of proteins in the fertilization envelope. However, by use of monoclonal and polyclonal antibodies to SFE1 and proteoliaisin, we show here that they are distinct gene products. We also show that SFE1 is packed selectively into the cortical granules and then is crosslinked into the fertilization envelope following fertilization. In situ RNA hybridization analysis shows that the mRNA of SFE1 (9 kilobases) is present in oocytes selectively and is turned over rapidly in the oocyte following germinal vesicle breakdown. Our findings suggest that the gene encoding this major product of the egg is activated concomitantly with the other cortical granule-specific products already identified, and that a common LDLr-like motif of the fertilization envelope may reveal a structural mechanism for protein interactions in its construction.

Direct molecular interaction of a conserved yolk granule protein in sea urchins.

The regulation of yolk storage in oocytes and subsequent utilization in embryos is critical for embryogenesis. In sea urchins, the major yolk protein is made in the intestines, transported to the ovaries and accumulated in developing oocytes within membrane-bound vesicles comprising approximately 10% of the mass of an egg. Here, a non-yolk protein that accumulates specifically in yolk granules is reported. This protein was identified by cDNA cloning and, by use of antibodies to the recombinant protein, it was shown that this molecule is stored selectively in yolk granules of oocytes and embryos. No accumulation was seen in the accessory cells, testis, or intestines. In situ ribonucleic acid (RNA) hybridizations showed that the transcript accumulated only in oocytes, and was more highly concentrated in young oocytes. However, later in oogenesis, the messenger ribonucleic acid (mRNA) levels decreased significantly so that no signal was detectable in mature haploid eggs or at any later stage in development. However, by immunofluorescence and western blot analysis, the 30 kDa band was present throughout development. The predicted sequence of this protein shows that it is a member of the bep, HLC-32, EBP family of sea urchin proteins, but as it does not accumulate at the cell surface, nor in the hyaline layer in the two species studied here, as do other members of the family, it has been referred to as YP30 (30 kDa protein of the yolk platelet). To address its potential function, yeast two-hybrid analysis was performed to screen for proteins that potentially interact with YP30. It was found that it binds itself, and forms strongly interacting dimers. It is hypothesized that YP30 participates in the packaging and storage of major yolk protein during oogenesis, or in the utilization of the major yolk protein in development.

A rab3 homolog in sea urchin functions in cell division.

Rabs are monomeric GTP binding proteins belonging to the ras superfamily that function throughout the secretory pathway. Members of the rab3 family function in the final steps of the secretory pathway, vesicle fusion with the plasma membrane. In contrast to mammalian systems with several rab3 isoforms (rab3A-D), a single family member homologue of rab3 is present in the rapidly dividing cleavage stage sea urchin embryo that localizes to numerous vesicles enriched at the cell cortex. We hypothesized that whereas the contents of these rab3-positive vesicles may contribute to the embryonic extracellular matrix, the membrane and its constituent proteins may be important for other aspects of cell division. We tested the function of rab3 in cell division by the microinjection of either antibodies or competing effector domain peptides to interfere with its function. We found that perturbing rab3 function results in cessation of cell division, whereas cells injected with either heat-inactivated antibodies or control scrambled peptides develop as normal. Moreover, neither endocytosis nor general membrane topology are affected by rab3 perturbation. Thus, we conclude that rab3-associated vesicles and/or their contents are critical for cell division.

SNAREs in mammalian sperm: possible implications for fertilization.

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.

The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations.

The acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus.

Syntaxin is required for cell division.

We recently identified a single family member homologue of syntaxin in the sea urchin. Syntaxin is present throughout development, and in rapidly dividing cleavage stage embryos it is present on numerous vesicles at the cell cortex. We hypothesized that syntaxin mediates essential membrane fusion events during early embryogenesis, reasoning that the vesicles and/or their contents are important for development. Here we show that functional inactivation of syntaxin with either Botulinum neurotoxin C1, which specifically proteolyzes syntaxin, or antibodies against syntaxin results in an inhibition of cell division. These observations suggest that syntaxin is essential for membrane fusion events critical for cell division.

How to grow a gut: ontogeny of the endoderm in the sea urchin embryo.

Gastrulation is the process of early development that reorganizes cells into the three fundamental tissue types of ectoderm, mesoderm, and endoderm. It is a coordinated series of morphogenetic and molecular changes that exemplify many developmental phenomena. In this review, we explore one of the classic developmental systems, the sea urchin embryo, where investigators from different backgrounds have converged on a common interest to study the origin, morphogenesis, and developmental regulation of the endoderm. The sea urchin embryo is remarkably plastic in its developmental potential, and the endoderm is especially instructive for its morphological and molecular responsiveness to inductive cell interactions. We start by examining and integrating the several models for the morphogenetic mechanisms of invagination and tissue elongation, the basic processes of endoderm morphogenesis in this embryo. We next critique the proposed mechanisms of inductive gene regulation in the endoderm that exemplifies a concept of modular transcriptional regulation. Finally, we end with an examination of the current molecular models to explain cell fate determination of the endoderm. Recent progress at the molecular level should soon allow us to explain the seminal experimental observations made in this embryo over a hundred years ago.

The cortical granule serine protease CGSP1 of the sea urchin, Strongylocentrotus purpuratus, is autocatalytic and contains a low-density lipoprotein receptor-like domain.

Trypsin-like activity is secreted from eggs of many species at fertilization, and this activity is believed to be critical for the block to polyspermy. Here we show that a cortical granule serine protease of sea urchins is the major and perhaps only protease family member important for fertilization. Zymography assays suggest that the cortical granules contain a single serine protease that can undergo autocatalysis and is secreted upon egg activation. We used this finding to identify a cDNA clone from a Strongylocentrotus purpuratus ovary cDNA library that encodes a 581-amino-acid-residue protein that we refer to as cortical granule serine protease 1 (CGSP1). The catalytic domain of the protein contains the essential residues of the catalytic triad characteristic of a member of the trypsin-like family of serine proteases and the N-terminus of CGSP1 resembles the ligand-binding domain of the low-density lipoprotein (LDL) receptor. Antibodies raised separately to both the protease and LDL receptor-like domains each localize to the cortical granules of unfertilized eggs. Furthermore, the full-length form of CGSP1, as well as intermediate and active forms of the protease, is detected in cortical granules by immunoblot analysis. Our evidence suggests that CGSP1 is activated at fertilization and is responsible for the protease-mediated reactions that follow cortical granule exocytosis and contribute to the block to polyspermy.

The TATA binding protein in the sea urchin embryo is maternally derived.

The cDNA encoding the TATA binding protein was isolated from 8- to 16-cell and morula-stage embryonic libraries of two distantly related species of sea urchin, Strongylocentrotus purpuratus and Lytechinus variegatus, respectively. The two proteins are 96% identical over both the N- and C-terminal domains, suggesting a conservation of transcriptional processes between the two species. The prevalence of SpTBP transcripts at several developmental time points was determined using the tracer excess titration method, and the corresponding number of TBP protein molecules was determined by quantitative Western blot analysis. Our results indicate that the amount of TBP mRNA and protein per embryo remains relatively constant throughout development. An initial large pool of TBP protein (>10(9)) molecules in the egg becomes diluted as a consequence of cell division and decreases to about 2 x 10(6) molecules per cell by the gastrula stage. We found by in situ RNA hybridization that the oocyte contains a large amount of TBP mRNA which is depleted late in oogenesis so that the eggs and early embryos have extremely low levels of TBP mRNA. We conclude that the oocyte manufactures nearly all of the TBP protein necessary for embryogenesis.

rab3 mediates cortical granule exocytosis in the sea urchin egg.

Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking.

A molecular analysis of hyalin--a substrate for cell adhesion in the hyaline layer of the sea urchin embryo.

The hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development. The major constituent of the hyaline layer is the protein hyalin, a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Here we provide a molecular characterization of hyalin and identify a region of the protein that is important for its function in cell adhesion. Partial hyalin cDNAs were identified from two sea urchin species, Strongylocentrotus purpuratus and Lytechinus variegatus, by screening expression libraries with monoclonal antibodies to hyalin. The cDNAs each encode a tandemly arranged series of conserved repeats averaging 84 amino acids. These hyalin repeats are as similar between the two species as they are to repeats within each species, suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences. The hyalin mRNA is approximately 12 kb in length and is present in developing oocytes coincident with the appearance of cortical granules, the vesicle in which the hyalin protein is specifically packaged. The mRNA is present throughout oogenesis but is rapidly lost at oocyte maturation so that eggs and early embryos have no detectable hyalin mRNA. The hyalin protein, however, remains at relatively constant levels throughout development. Thus, all the hyalin protein present during early development, when no RNA is detectable, is maternally derived and exocytosed from cortical granules at fertilization. Hyalin mRNA reaccumulates in embryos beginning at the mesenchyme blastula stage; a RNA gel blot and in situ hybridization analysis of gastrulae and larvae shows a progressive confinement of hyalin mRNA to the aboral ectoderm. Recombinant hyalin containing the tandem repeat region of the protein was expressed in bacteria and is shown to serve as an adhesive substrate, almost equal to that of native hyalin, in cell adhesion assays. This adhesive activity is partially blocked by dilute hyalin monoclonal antibody Tg-HYL to the same extent as that for native hyalin. Thus, this hyalin repeat region appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development and now enable investigators to focus on the mechanisms of cell interactions with the hyaline layer.

Sea urchin ovoperoxidase: oocyte-specific member of a heme-dependent peroxidase superfamily that functions in the block to polyspermy.

Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin cortical granules, released during the cortical reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S. purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization reactions of other metazoan eggs.