Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

Searching for candidate speciation genes using a proteomic approach: seminal proteins in field crickets

In many animals, male seminal proteins influence gamete interactions and fertilization ability and are probably involved in barriers to gene flow between diverging lineages. Here we use a proteomic approach to identify seminal proteins that are transferred to females during copulation and that may be involved in fertilization barriers between two hybridizing field crickets (Gryllus firmus and Gryllus pennsylvanicus). Analyses of patterns of divergence suggest that much of the field cricket genome has remained undifferentiated following the evolution of reproductive isolation. By contrast, seminal protein genes are highly differentiated. Tests of selection reveal that positive selection is likely to be responsible for patterns of differentiation. Together, our observations suggest that some of the loci encoding seminal proteins may indeed play a role in fertilization barriers in field crickets.     

Expression and segregation of nucleoplasmin during development in Xenopus

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.     

Protein Labelling Patterns in Oocytes of Xenopus laevis

Soluble and microsomal proteins synthesized at various stages of oogenesis in Xenopus laevis were compared by ³H and ¹⁴C dual-isotope labelling with subsequent mixing and analysis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results indicated that the proteins labelled in all the stages of oogenesis studied are remarkably similar. However, the patterns in the small and medium-sized oocytes are more similar to one another than to the patterns characteristic of large oocytes. The greatest differences were found when comparing the microsomal proteins.
The labelling patterns of oocyte proteins in vitro were not significantly different from the in vivo patterns for the stages of oogenesis studied. These results indicate that (1) there is little quantitative contribution of proteins to either the soluble or microsomal fractions from extra-oocytic sources in vivo and (2) the in vitro system itself has little effect on the labelling patterns over the incubation period.     

Ultrastructural Observations on Fertilization in Dionchus remorae (Platyhelminthes,Monogenea, Dionchidae)

Ultrastructural observations are presented for some of the stages occurring during fertilization in Dionchus remorae (a gill parasite of Echeneis naucrates) and are believed to be the first published concerning a monogenean. Fertilized female germ cells were found in the ovary. Several loops of the spermatozoon were present within the oocyte cytoplasm; the sperm nucleus became electron lucent and the parallel peripheral doublets of the axonemes became increasingly divergent. The cortical granules in the oocyte were not released immediately after penetration by the spermatozoon. The homogeneity apparently found in the oocyte ultrastructure and process of fertilization in the monogeneans and digeneans contrasts with the variety that exists in their sperm ultrastructure.     

Two-dimensional gel analysis of the fate of oocyte nuclear proteins in the development of Xenopus laevis

The amphibian oocyte nucleus is thought to provide a maternal store of protein required in embryogenesis. The fate of germinal vesicle proteins has been studied by comparing polypeptide patterns of oocytes, embryos, and several adult organs of Xenopus laevis on two-dimensional gels. A combination of silver staining and fluorography of radiolabeled protein on gels was used to analyze maternal and newly synthesized polypeptides in embryogenesis. Comparison of protein patterns was facilitated and corroborated by application of monoclonal antibodies against several germinal vesicle proteins. These were characterized by immunoblotting from two-dimensional gels, and polypeptides of identical structure were recognized in oocyte nuclei, embryos, and tadpoles. The following conclusions were drawn: (1) Almost all prevalent germinal vesicle proteins can be continuously traced in embryos up to swimming tadpole stages, although their patterns of new synthesis are greatly different, some are not radiolabeled in the embryo but solely provided by the maternal store. (2) Many of the polypeptides occurring in oocyte nuclei are also found in one or several organs of the adult. (3) Tissue specificities of germinal vesicle proteins, previously detected by immunocytochemistry with monoclonal antibodies, could be confirmed by independent biochemical methods. (4) As has been previously shown by immunohistological methods, oocyte nuclear antigens are shed into the cytoplasm of the maturing egg, and are reaccumulated in the nuclei of the embryonic cells, each at a characteristic developmental stage. These shifts between intracellular compartments are not accompanied by a change of the covalent structure of the antigen.     

Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes,zygotes, and pre-implantation stage embryos

Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature oocytes and decline after fertilization, suggest that GSH synthesis may be associated with cell cycle events. To explore this possibility, we measured the concentrations of GSH in Golden Hamster oocytes and zygotes at specific stages of oocyte maturation and at intervals during the first complete embryonic cell cycle. Between 2 and 4 hr after the hormonal induction of oocyte maturation, GSH concentrations increased significantly (approximately doubling) in both oocytes and their associated cumulus cells. This increase was concurrent with germinal vesicle breakdown and the condensation of metaphase I chromosomes in the oocyte. GSH remained high in ovulated, metaphase II (MII) oocytes, but then declined significantly, by about 50%, shortly after fertilization, as the zygote progressed back into interphase (the pronucleus stage). GSH concentrations then plummeted by the two-cell embryo stage and remained at only 10% of those in MII oocytes throughout pre-implantation development. These results demonstrate that oocyte GSH concentrations fluctuate with the cell cycle, being highest during meiotic metaphase, the critical period for spindle growth and development and for sperm chromatin remodeling. These observations raise the possibility that GSH synthesis in maturing oocytes is regulated by gonadotropins, and suggest that GSH is more important during fertilization than during pre-implantation embryo development.     

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.     

Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals

Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenopus laevis larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.     

Acid Release during Activation of Barnea candida (Mollusca, Pelecypoda) Oocytes

Barnea caridida oocytes release acid (1.35 pmole H⁺/oocyte) upon fertilization. After artificial activation by an excess of KCl, germinal vesicle breakdown (GVBD) occurs normally and a quite similar, but not identical, acid release is recorded (1.10 pmole H⁺/oocyte). KCl activation of Barnea oocytes is completely inhibited in 100 mM sodium-acetate sea water at pH 6.5 and fertilization does not result in activation when the oocytes are transferred after one minute into 100 mM sodium-acetate sea water at pH 6.3. When D–600, a calcium transmembrane fluxes inhibitor, is added 20 seconds after fertilization, GVBD is inhibited but a normal acid release is recorded. The presence of at least 10 mM sodium ions in the external medium is required for 100% activation of these oocytes by an excess of KCl. These results suggest that while an intracellular pH increase may be a requisite for GVBD, this can not be a sufficient condition to trigger it unless a calcium influx is allowed to occur. Moreover, the acid release does not result from a Ca⁺⁺-H⁺ exchange transport but appears more likely to be due to a Na⁺-H^* exchange as it has been demontrated in sea urchin eggs.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

Dynamic changes to the inositol 1,4,5-trisphosphate and ryanodine receptors during maturation of bovine oocytes

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR) have been identified as two ligand-gated calcium channels which play a critical role in mediating calcium release in many different types of cells and tissues. The physiological significance of the two receptors in regulation of intracellular calcium during meiotic maturation and fertilization in the bovine oocyte was evaluated. Metabolic labeling of bovine oocytes by Met-Cys 35S during early and late maturation was followed by immunoprecipitation of both RyR and IP3R using specific antibodies against these two receptors. Results indicate that IP3R is translated throughout the maturation period; in contrast, RyR is only translated during the late maturation period of bovine oocytes. In addition, the experiments reported here investigate the temporal and spatial relationships between these calcium channels and the endoplasmic reticulum (ER) and cortical granules (CG). Immunocytochemistry, fluorescence staining and confocal microscopy were applied at four oocyte developmental stages: the germinal vesicleintact (GV-intact), metaphase I (MI) and metaphase II (MII) stages of maturation and the fertilized egg at 6 h post insemination (hpi). Although oocytes demonstrated some differences in staining patterns and localization, both receptor types showed apparent dynamic changes during meiotic maturation and dramatic decreases in signals after insemination. These results indicate the changes in the number and distribution of IP3R and RyR may account for the increased intracellular calcium responsiveness at fertilization. The IP3R appears to associate with the ER at the sub-vitelline membrane cortex in bovine oocytes. In addition, RyR appears to associate with the CG. In conclusion, although these two receptors may have different functional roles in regulation of calcium release during meiotic maturation and fertilization, it appears that both IP3R and RyR contribute to the significant increase of intracellular calcium during fertilization and activation in the bovine oocyte.     

Developmental capability of denuded bovine oocyte in a co-culture system with intact cumulus-oocyte complexes: role of cumulus cells, cyclic adenosine 3',5'-monophosphate, and glutathione

Cumulus oophorus cells have been implicated in the regulation of female gamete development, meiotic maturation, and oocyte-sperm interaction. Nevertheless, the specific role of cumulus cells (CCs) during the final stages of oocyte maturation and fertilization processes still remains unclear. Several studies have been conducted in order to clarify the role of follicular cells using culture systems where denuded oocytes (DOs) were co-cultured with isolated CCs, or in the presence of conditioned medium. However, those attempts were ineffective and the initial oocyte competence to become a blastocyst after fertilization was only partially restored. Aim of the present study was to analyze the effect of the interactions between somatic cells and the female gamete on denuded oocyte developmental capability using a system of culture where CCs were present as dispersed CCs or as intact cumulus-oocyte complexes (COCs) in co-culture with oocytes freed of CC investment immediately after isolation from the ovary. Moreover, we analyzed the specific role of cyclic adenosine 3'-5' monophosphate (cAMP) and glutathione (GSH) during FSH-stimulated maturation of denuded oocyte co-cultured with intact COCs. Our data confirm that denuded oocyte has a scarce developmental capability, and the presence of dispersed CCs during in vitro maturation (IVM) does not improve their developmental competence. On the contrary, the co-presence of intact COCs during denuded oocyte IVM partially restores their developmental capability. The absence of CCs investment causes a drop of cAMP content in DOs at the beginning of IVM and the addition of a cAMP analog in the culture medium does not restore the initial oocyte developmental competence. The relative GSH content of denuded oocyte matured in presence of intact COCs is consistent with the partial recovery of their developmental capability. However, the complete restoration of a full embryonic developmental potential is achieved only when DOs are co-cultured with intact COCs during both IVM and in vitro fertilization (IVF). Our results suggest that the direct interaction between oocyte and CCs is not essential during IVM and IVF of denuded oocyte. We hypothesize that putative diffusible factor(s), produced by CCs and/or by the crosstalk between oocyte and CCs in the intact complex, could play a key role in the acquisition of developmental competence of the denuded female gamete.