Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Comparative proteome analysis of changes in the 26S proteasome during oocyte maturation in goldfish

Proteasomes are large, multi-subunit particles that act as the proteolytic machinery for most of the regulated intracellular protein degradation in eukaryotic cells. An alteration of proteasome function may be important for the regulation of the meiotic cell cycle. To study the change at the subunit level of the 26S proteasome during meiotic maturation, we purified 26S proteasomes from immature and mature oocytes of goldfish. Two-dimensional polyacrylamide gel electrophoresis was used to separate proteins. For differential analysis, whole spots of the 26S proteasome from goldfish oocytes were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database analysis. Four spots that were different (only detected in mature oocyte 265 proteasomes and not in immature ones) and four protein spots that were up- or down-regulated were identified unambiguously. The mature-specific spots were not 26S proteasome components but rather their interacting proteins, and were identified as chaperonin-containing TCP-1 subunits and myosin light chain. Minor spots of three subunits of the 20S core particle and one of the 19S regulatory particle showed meiotic cell cycle-dependent changes. These results demonstrate that modifications of proteasomal subunits and cell cycle phase-dependent interactions of proteins with proteasomes occur during oocyte maturation in goldfish.     

Temporal pattern of synthesis of the mouse cortical granule protein, p75, during oocyte growth and maturation.

We previously demonstrated that a protein of M(r) 75,000 (p75) is localized to cortical granules (CGs) in mouse oocytes and eggs and is released upon activation or fertilization of eggs (K.E. Pierce, M. C. Siebert, G. S. Kopf, R. M. Schultz, and P. G. Calarco, 1990, Dev. Biol. 141, 381-392). To examine the temporal pattern of synthesis of p75 during the early stages of CG formation, growing oocytes, which were isolated from juvenile mice, were incubated for 4 hr in medium containing [35S]methionine, and radiolabeled proteins were immunoprecipitated using an antiserum that detects p75. Synthesis of p75 is detected at low levels in the smallest oocytes examined (less than 20 microns). Synthesis of p75 relative to total protein synthesis increases about 12-fold during oocyte growth from the 20-40 microns size and then remains constant throughout the remaining period of oocyte growth (40-70 microns). In the fully grown, germinal vesicle (GV)-intact oocyte (70-80 microns), immunoprecipitated p75 comprises approximately 1.5% of the total amount of radiolabeled protein. Three hours after the transfer of these oocytes to a medium that supports resumption of meiosis and GV breakdown in vitro, oocytes subjected to a 1-hr labeling pulse display a 35% decrease in the relative level of p75 synthesis. By 15 hr of maturation, p75 synthesis was reduced to 14% of that in the fully grown, GV-intact oocyte and this is similar to the level of p75 synthesis in ovulated eggs. The level of p75 synthesis following in vitro translation of total egg RNA is only 38% lower than that obtained from total oocyte RNA. In addition, synthesis of p75 is observed following in vitro translation of oocyte, but not egg, poly(A)+ RNA. These results are consistent with p75 synthesis during oocyte maturation being under translational control.     

Regulatory contribution of heterotrimeric G-proteins to oocyte maturation in the sea urchin

Regulation of animal oocyte maturation is hypothesized to involve heterotrimeric G-proteins. It is difficult to test this hypothesis though without knowing what G-proteins are present in these cells and where are they localized. We set out to test the hypothesis that G-proteins regulate maturation in the sea urchin oocyte by identifying resident G-proteins in oocytes and eggs, and then investigating their function. We find four families of G-protein alpha-subunits (Galphai, Galphaq, Galphas, and Galpha12) present in both oocytes and eggs of the sea urchin. Three of them, Galphai, Galphaq, and Galphas are present on the plasma membrane of the oocyte, while the fourth is located on cytoplasmic vesicles. Upon oocyte maturation, these proteins remain in eggs, and continue to be expressed in embryonic tissues. To test the functional contribution of the G-proteins to the regulation of oocyte maturation, we employ specific intervening reagents, including antibodies and competitor peptides to each Galpha subunit, and specific Galpha toxins. We find that Gi is a main candidate for a positive regulator of sea urchin oocyte maturation. These studies provide a foundation to further test specific hypotheses of the G-protein mediated regulation of oocyte maturation, fertilization, and early development in the sea urchin.     

Talin and vinculin in the oocytes, eggs, and early embryos of Xenopus laevis: a developmentally regulated change in distribution

We have investigated the expression and distribution of talin and vinculin in the oocytes, eggs, and embryos of Xenopus laevis. Antibodies to the previously characterized avian proteins stain several different Xenopus cell types identically by immunofluorescence: adhesion plaques of cultured kidney (A6) cells, the cell peripheries of oviduct cells, and the postsynaptic neuromuscular junctions of tadpole tail muscle fibers. These antibodies also identify cognate proteins of the appropriate sizes on immunoblots of A6 cell and oviduct lysates. Using these antibodies on ovarian tissue, we find talin to be highly localized at the cortices of oocytes and vinculin to be in the oocyte cytoplasm and absent from the oocyte cortex. In the cells of the ovarian layers that surround the oocytes, talin and vinculin can be detected as soluble and cytoskeletal components. Vinculin is first detectable as a cytoskeletal component in eggs, appearing some time during or between oocyte maturation and oviposition. During early embryo development, talin and vinculin are colocalized in the cortex of cleavage furrows and blastomeres. Thus, Xenopus oocytes and eggs display different distributions of talin and vinculin. The change from unlinked localization to colocalization appears to be developmentally regulated, occurring during the transition from oocyte to egg.     

DFP-sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad,Bufo japonicus

The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0–0.5 GVBD₅₀ on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD₅₀) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process. These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo. © 1994 Wiley-Liss, Inc.     

Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

p70(S6K) controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis

In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.     

Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.

Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.     

The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans

Summary Externally applied membrane permeable cAMP derivatives and the injection of cAMP induce oocyte maturation in several species of hydrozoans. This technique for inducing oocyte maturation has been used to study ion permeability changes, maturation promoting factor activity and surface tension changes during maturation. Oocyte membrane potential remains constant during maturation. Cyclic AMP induced maturation proceeds in the absence of external Ca²⁺, K⁻, Mg²⁺ or Na⁺. Cytoplasm from maturing oocytes that induces oocyte maturation when it is injected into untreated oocytes is produced during cAMP induced maturation. Surface tension, as measured by the application of a standardized force that mechanically deforms individual oocytes, declines during the first part of maturation. This is followed by a sharp rise and fall of surface tension at first and second polar body formation that accompanies a slow rise in the resistance of oocytes to deformation during the last part of maturation. The production of maturation promoting factor activity and some of the changes in surface tension during maturation can occur in the absence of germinal vesicle material. Two early developmental events that follow oocyte maturation are the production of sperm chemoattractant and calcium channel function. Neither of these events occurs in eggs that have undergone maturation in the absence of germinal vesicle material. The addition of germinal vesicle contents from oocytes to eggs that have undergone maturation in the absence of germinal vesicle material initiates calcium channel function. This experiment indicates that the germinal vesicle contains factors that are necessary for post-maturation developmental events.     

Essential role of ubiquitin C-terminal hydrolases UCHL1 and UCHL3 in mammalian oocyte maturation

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of deubiquitinating enzymes that play a role in the removal of multi-ubiquitin chains from proteins that are posttranslationally modified by ubiquitination to be targeted for proteolysis by the 26S proteasome. The UCH-enzymes also generate free monomeric ubiquitin from precursor multi-ubiquitin chains and, in some instances, may rescue ubiquitinated proteins from degradation. This study examined the roles of two oocyte-expressed UCHs, UCHL1, and UCHL3 in murine and rhesus monkey oocyte maturation. The Uchl1 and Uchl3 mRNAs were highly expressed in GV and MII oocytes, and were associated with the oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Microinjection of the UCH-family enzyme inhibitor, ubiquitin-aldehyde (UBAL) to GV oocytes prevented oocyte meiotic progression beyond metaphase I in a majority of treated oocytes and caused spindle and first polar body anomalies. Injection of antibodies against UCHL3 disrupted oocyte maturation and caused meiotic anomalies, including abnormally long meiotic spindles. A selective, cell permeant inhibitor of UCHL3, 4, 5, 6, 7-tetrachloroidan-1, 3-dione also caused meiotic defects and chromosome misalignment. Cortical granule localization in the oocyte cortex was disrupted by UBAL injected after oocyte maturation. We conclude that the activity of oocyte UCHs contributes to oocyte maturation by regulating the oocyte cortex and meiotic spindle.     

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.     

Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

In order to gain further insight into IGF-1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF-1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52(ShcA). Western blot analysis using homologous antibodies evidenced a 60-kDa protein, p60(Xl)(Shc), that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60(Xl)(Shc), Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant-negative form of Grb2 specifically inhibits insulin-induced resumption of meiosis. We finally show that Grb2 binds to p60(Shc) in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2-Sos are implicated in ras-dependent Xenopus oocyte maturation induced by insulin/IGF-1; they also indicate that inability of insulin/IGF-1 to activate the Ras-MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

Treatment with the Proteasome Inhibitor MG132 during the End of Oocyte Maturation Improves Oocyte Competence for Development after Fertilization in Cattle

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 16–22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM – effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16–22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.     

Oocyte aging in a marine protostome worm: The roles of maturation‐promoting factor and extracellular signal regulated kinase form of mitogen‐activated protein kinase

The roles of maturation‐promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen‐activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase‐I‐arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho‐specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation‐initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.