Regulation of simian virus 40 gene expression in Xenopus laevis oocytes.

Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand-specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E-strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated.     

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.     

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.     

Characterization and localization of a mouse egg cortical granule antigen prior to and following fertilization or egg activation.

Immunological approaches were used to characterize an antigen that is present within the cortical granules of mouse oocytes and eggs. Immunoelectron microscopy shows a specific localization of the antigen to the cortical granules in the cortex of mouse oocytes and eggs. Following in vitro fertilization, the antigen is present in the perivitelline space and is associated with the zona pellucida. No cortical granules and very little antigen are detected in the two-cell embryo. This antiserum detects a protein of Mr = 75,000 (p75) following immunostaining of egg proteins on Western blots, or immunoprecipitation of metabolically labeled oocyte proteins or radio-iodinated egg proteins. p75 is also present in exudates obtained from A23187-treated eggs, as detected by either radio-iodination of the released egg proteins, or maturation and ionophore activation of metabolically labeled oocytes. Two-dimensional gel electrophoresis of radio-iodinated egg proteins reveals four species of p75 with pIs between 4.9 and 5.3, whereas only the most basic form of p75 is detected in metabolically labeled oocytes. Multiple forms of the radio-iodinated p75 are present in the exudate of ionophore-treated eggs. p75 displays a greater electrophoretic mobility under nonreducing conditions, indicating the presence of intramolecular disulfide bonds, a common characteristic of secreted proteins. We conclude that p75 is synthesized in oocytes, modified and packaged into cortical granules, and released from eggs following fertilization or activation.     

Analysis of the Formation of the Animal-Vegetal Axis during Xenopus Oogenesis Using Monoclonal Antibodies

Maternally accumulated materials in Xenopus oocytes, in particular mRNAs and proteins, are considered to participate in the determination of the developmental specification of embryonic cells. In this study, a large number of monoclonal antibodies was raised against bulk oocyte antigens to examine patterns of intracellular distribution of oocyte proteins. Immunohistochemical experiments with mature oocytes showed that there are five different patterns of distribution of oocyte proteins, with enrichment on the animal side (type A₁, and A₂ ptoteins), vegetal side (type V₁ and V₂ proteins), and in the peripheral cytoplasm (type P proteins). Clear localization of type A and V antigen proteins occurred at Dumont's stages IV-VI. However, at the preceding stages, the distributions of these antigen proteins appeared to be homogeneous. By contrast, the pattern of distribution of type P protenis did not change markedly throughout oogenesis. The presence of type A and type V antigen proteins reflected the animal-vegetal axis in the cytoplasm of the mature oocyte. Furthermore, there were two boundaries of the distributions of proteins at the equatorial region, excluding or including the cytoplasm around the germinal vesicle. Thus, the cytoplasm of mature oocytes was multilayered with respect to the different proteins distributed along the animal-vegetal axis.     

Protein synthesis in oocytes of Xenopus laevis is not regulated by the supply of messenger RNA

The control of protein synthesis in oocytes of Xenopus laevis has been investigated by injecting oocytes with mRNA and polysomes followed by labeling with ¹⁴C-amino acid mixtures. Contrary to previous reports in which injected oocytes were labeled with ³H-histidine, injected globin mRNA is found to decrease amino acid incorporation into endogenous proteins competitively at all concentrations tested. No increase in overall amino acid incorporation is detected when more mRNA is supplied. Similar results are obtained after labeling injected oocytes with leucine, methionine, proline or valine individually. An explanation is presented for the conflicting results obtained when histidine is used as a label.When reticulocyte polysomes are injected, rather than purified globin mRNA, incorporation of amino acids into endogenous proteins remains roughly constant and overall incorporation increases. Similarly, when encephalomyocarditis viral RNA is injected together with either globin mRNA or reticulocyte polysomes, the globin mRNA causes decreased amino acid incorporation into encephalomyocarditis proteins, but the polysomes do not do so. The results demonstrate that different types of mRNA compete for a strictly limited translational capacity which is saturated in the normal oocyte. The limiting component is present in polysomes and is not message-specific. The constraint on protein synthesis in the amphibian oocyte cannot be fully explained by masked mRNA.     

Immunological identification of the karyophilic, histone-binding proteins N1 and N2 in somatic cells and oocytes of diverse amphibia

A polypeptide pair designated N1/N2 (Mr 100 000 and 110 000) is an exceptionally acidic and abundant nuclear protein of oocytes of the toad, Xenopus laevis, and is characterized by a pronounced karyophilia. These proteins have been shown to form specific complexes with free, i.e., non-chromatin-bound histones H3 and H4 (Kleinschmidt & Franke, Cell 29 (1982) 799) [3]. In order to study these proteins and their possible counterparts in other species, antibodies were produced in guinea pigs against proteins N1/N2 purified from Xenopus oocyte nuclei. Using gel electrophoresis, peptide map analysis, immunoblotting techniques and immuno fluorescence microscopy the existence of polypeptides identical in Mr value and charge to polypeptide N1 of oocytes was demonstrated in cultured somatic cells of Xenopus laevis, where it was also highly enriched in cell nuclei, although the cellular concentration was much lower than in oocytes. A similar, if not identical protein, was recognized in nuclei of diverse other cell types including hepatocytes, enterocytes, ovarian follicle cells, and Sertoli cells of testis, of Xenopus, Rana temporaria, R. esculenta, Pleurodeles waltlii but not in erythrocytes and later stages of spermiogenesis. When nuclear proteins from oocytes of different amphibian species were examined with these antibodies it was found that the Mr values of N1/N2 proteins were considerably different in different species, ranging from Mr 110 000 to 190 000. Immunoprecipitation and gel electrophoretic analysis under non-denaturing conditions showed that a significant proportion of these proteins was contained in complexes with histones H3 and H4. The results demonstrate that proteins N1/N2 are not special proteins of oocytes of Xenopus laevis but occur in various other cells of diverse amphibian species. The widespread occurrence of these karyophilic proteins indicates that at least one function of these proteins, i.e., selective binding of the arginine-rich histones H3 and H4, is not exclusive to oocytes but may also contribute to the regulation of histone pools and chromatin formation in other cell types.     

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.     

Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression.

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.     

The role of germinal vesicle in maturation of Pleurodeles waltlii oocytes induced by steroids

The maturation of oocytes from unstimulated Pleurodeles waltlii females can be induced in vitro. All full-grown oocytes covered by follicular envelopes mature under the influence of progesterone, testosterone, and a synthetic steroid (CDMT). Enucleation of oocytes prior to maturation induction resulted in a significantly lower percentage of maturation. Irradiation, or the treatment of oocytes with actinomycin D, did not affect their maturation. Therefore the lower percentage of maturation after enucleation must be due to the lack of karyoplasm. Karyoplasm appears to be necessary for the production of the maturation-promoting factor in P. waltlii oocytes.     

Hepatitis B core and surface antigens and delta agent in chronic liver disease in Kuwait

Hepatitis B surface antigen was detected immunohistochemically in 25 out of 85 liver biopsies (29.4%) of chronic liver disease. Core antigen was also demonstrated in 9 of the 25 Hepatitis HBs Ag positive biopsies (36%). Delta agent however, was found in only one case of HBs positive chronic active hepatitis. The number of hepatocytes staining positively for HBc antigen was greater in those biopsies with the strongest staining for HBs antigen. The only case of chronic active hepatitis positive for delta agent showed that the positive staining was confined to the nuclei of few hepatocytes. The routine histology showed chronic active hepatitis with a moderate degree of inflammation. The present results confirm our previous reports that almost one third of chronic liver disease in Kuwait is associated with hepatitis B infection. Previous serological studies suggest that delta agent infection is also common; however, the present study suggest that delta agent may be a transient, and not a major, contributing factor in the progression of liver disease.     

Analysis of vitelline envelope synthesis and composition during early oocyte development in gilthead seabream (Sparus aurata)

The oocyte vitelline envelope (VE) of gilthead seabream is composed of four known zona pellucida (ZP) proteins, ZPBa, ZPBb, ZPC, and ZPX. We have previously shown that the gilthead seabream ZP proteins are differentially transcribed in liver and ovary, with the expression in liver being under estrogenic control. However, although mRNA was found in both liver and ovary, only low ZPBa protein levels were detected in liver and plasma. Using isoform-specific ZP antibodies we show that ZPBa and ZPX translation products are present in the cytosol of stage I and II oocytes. In addition, the zpBa and zpX mRNAs were detected in early developing oocytes. During oocyte growth (vitellogenesis), the VE increased in thickness (>10 microm), and we show that the four ZP isoforms are present in different regions of the VE. ZPX was detected closest to the oocyte plasma membrane while the intermediate region was composed of ZPBa, ZPBb, and ZPC. At the outer layer, only ZPC was detected. When oocytes reach the fully grown stage they resume meiosis and hydration. As the oocyte expands, thinning to 4 microm, the VE acquire a striped and compact appearance at the electron microscopy level. This study provides further evidence for the oocyte origin of some ZP proteins in the gilthead seabream and suggests that the ZP proteins are differentially distributed within the VE.     

Characterization of Unusual Escape Variants of Hepatitis B Virus Isolated from a Hepatitis B Surface Antigen-Negative Subject

Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee’s sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.     

Centrifugal bisection of starfish oocytes

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.     

Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl

Summary The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.