Microtubule protein synthesis during oogenesis and early embryogenesis in Xenopus laevis.

A method is described which permits the preparation of descrete classes of oocytes of different sizes from all stages of oogenesis in Xenopus laevis. This technique is used in the determination of the content of microtubule protein in oocytes during the course of oogenesis. These experiments show that microtubule protein is present in oocytes of all sizes assayed and that the amount is simply related to the volume of the oocyte. In the largest oocytes microtubule protein constitutes 1% of the soluble protein and this amount does not change on maturation and fertilization. These results show that the changes occurring in the oocyte on maturation which allow the cytoplasm to support microtubule polymerization occur as a result of a modification of the pre-existing microtubule protein, not from protein synthesis de novo. These experiments also indicate that the synthesis of microtubule protein either form 'masked' mRNA or from newly synthesized mRNA plays an insignificant role in microtubule protein synthesis at maturation, ovulation and immediately post-fertilization.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.     

The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

Identification and expression analysis of GPAT family genes during early development of Xenopus laevis

Production of lysophosphatidic acid (LPA) is the first step in the de novo pathway for glycerolipid biosynthesis, which is mainly catalyzed by the glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15). DHAPAT (EC2.3.1.42) also contributes in a minor way, using dihydroxyacetone phosphate as substrate. Final products and intermediates of the glycerolipid synthesis pathway are the main structural components of cellular membranes, and provide signalling molecules that regulate diverse biological processes, including cell proliferation, differentiation and growth. Here we identified the four orthologs of the mammalian GPATs (1-4) and DHAPAT in Xenopus, including a novel, short variant of GPAT2, and analyzed their expression pattern during embryonic development. Xenopus GPAT1/2 localized to mitochondria, while GPAT3/4 associated with the endoplasmic reticulum. All are similarly expressed in the early embryonic nervous system. A more tissue specific pattern emerges during organogenesis, including liver expression for GPAT1/4, and testis expression for GPAT2. All acyltransferases were expressed in kidney, though GPAT3 was excluded from the pronephric ducts. Our results suggest important roles of GPATs and DHAPAT during early organogenesis.     

Role of TSC-22 during early embryogenesis in Xenopus laevis

Transforming growth factor-beta1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved. During mouse embryogenesis, TSC-22 is expressed at the site of epithelial-mesenchymal interaction. Here, we isolated Xenopus laevis TSC-22 (XTSC-22) and analyzed its function in early development. XTSC-22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC-22 antisense morpholino oligonucleotides (XTSC-22-MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent-extension, as confirmed by whole-mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC-22-depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC-22 mRNA-injected embryos. Co-injection of XTSC-22-MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC-22 is required for cell movement during gastrulation though cell cycle regulation.     

Cloning and developmental expression of WSTF during Xenopus laevis embryogenesis

The gene WSTF is deleted in the autosomal dominant hereditary disorder Williams-Beuren syndrome. This disorder is caused by a 1.3 megabase deletion in human chromosome 7, encompassing at least 17 genes. The WSTF protein contains a bromodomain, found predominantly in chromatin-associated proteins. Reported association of WSTF with chromatin remodeling factors and functional data support a role for WSTF during chromatin remodeling. Here, we report the cloning and developmental expression pattern of Xenopus laevis WSTF. Xenopus laevis WSTF is a protein with a predicted amino acid sequence of 1441 amino acids. Three discrete domains can be identified in the Xenopus laevis WSTF protein, a PHD finger, a DDT domain and a bromodomain. Alignment of Xenopus WSTF with the corresponding orthologues from Homo sapiens, Gallus gallus, Mus musculus and Danio rerio demonstrates an evolutionary conservation of WSTF amino acid sequence and domain organization. In situ hybridization reveals a dynamic expression profile during embryonic development. WSTF is expressed differentially in neural tissue, especially during neurulae stages in the eye, in neural crest cells and the brain.     

Cell cycle control and early embryogenesis: Xenopus laevis maturation and early embryonic cell cycles

Our understanding of how meiotic maturation is regulated in Xenopus laevis continues to flourish. Premature initiation of maturation is prevented by the cAMP-dependent protein kinase, which inhibits the synthesis of Mos and potently blocks activation of cdc25. The autoamplification of maturation promoting factor (MPF) activity can be explained by the ability of MPF to directly activate cdc25. Later, in Meiosis II, the contribution of Mos to cytostatic factor (CSF) appears to be mediated through its activation of the mitogen-activated protein kinase, and cdk2 has been added to the active components of CSF. A model is presented illustrating the pathways of meiotic reinitiation, and indicating gaps in our knowledge.     

Changes in nuclear localization of An3, a RNA helicase,during oogenesis and embryogenesis in Xenopus laevis

The immunolocalization of An3 protein, an ATP-dependent RNA helicase and a member of the DEAD box family, was compared with the localization of fibrillarin, a protein essential for rRNA processing, and snRNPs, which are involved in mRNA splicing reactions, during oogenesis and embryogenesis in Xenopus laevis. Although An3 protein was detected in the cytoplasm of all stages of oocytes, in most stages An3 protein was also present in the nucleus. Prior to stage I An3 protein was uniformly dispersed throughout the entire germinal vesicle; from stages I to V it was in nucleoli. By stage VI nucleolar labeling with anti An3 disappeared and the protein was no longer present within nuclei. An3 reactivity was also present throughout the nuclei of follicle cells surrounding prestage I to stage VI oocytes. Both cytoplasmic and nuclear An3 staining were present in cells of stages 8 to 35 embryos; however, nuclear staining was punctate and uniformly distributed throughout the nucleoplasm. Fibrillarin was diffusely distributed throughout the entire germinal vesicle prior to stage I, localized exclusively to nucleoli of oocytes between stages I and VI and in nucleoli of stages 12 and 35 embryonic cells. Reactivity for snRNPs (anti-Sm) in germinal vesicles of prestage I oocytes was diffuse, and similar to the distribution of An3 and fibrillarin; in later stage oocytes anti-Sm staining was restricted to a population of granules, much fewer in number and more heterogeneous in size than nucleoli. Anti-Sm activity was apparent in nuclei of embryonic cells of stages 8 to 35 embryos. Although colocalization of the Sm epitope and An3 was not observed in developing oocytes and in embryonic cells, Sm reactive material was frequently found in close association with An3-positive nucleoli (oocytes) and nuclear deposits (embryonic cells). In stage IV and V oocytes treated with actinomycin D (4 μg/ml) to inhibit rRNA synthesis, nucleoli, which continued to possess fibrillarin, lacked An3; staining of follicle cell nuclei for An3 was unchanged. Treatment with 200 μg/ml actinomycin D to block mRNA synthesis, inhibited An3 but not fibrillarin staining in nuclei of prestage I oocytes and follicle cells. The changing patterns of An3 reactivity and the differential effects of actinomycin D on such localizations observed here are consistent with a role for An3 in the processing/production of RNA. © 1996 Wiley-Liss, Inc.     

Characterization of the O-GlcNAc protein modification in Xenopus laevis oocyte during oogenesis and progesterone-stimulated maturation

Little information exists about single N-acetylglucosamine modifications on proteins in growth and developmental model systems. To explore these phenomena, Xenopus laevis oocytes from stages I-VI of oogenesis were isolated and proteins analyzed on SDS-PAGE. The proteins were probed with antibodies specific for O-GlcNAc. Levels of the O-GlcNAc protein modification were highest in stages I and II, while decreasing in stages III-VI. The reduction in amount of O-GlcNAc-modified proteins was correlated to increases in apparent O-GlcNAcase (streptozotocin-inhibitable neutral hexosaminidase), activity involved in removing protein monoglycosylations. The O-GlcNAc modification was also characterized during progesterone-stimulated oocyte maturation. Although O-GlcNAcase activity appeared relatively constant between quiescent and matured stage VI oocytes, a small decrease in the levels of both total and specific O-GlcNAc-modified proteins was observed. Investigating the function of O-GlcNAc during maturation, oocytes were incubated with compounds known to modulate the levels of the O-GlcNAc protein modification and then stimulated to mature. Oocytes treated with compounds known to increase O-glycosylation consistently matured slower than non-treated controls, while oocytes treated with compounds that decrease O-glycosylation matured slightly faster than controls. The O-GlcNAc modification may play important roles in both the developmental and cell division processes of X. laevis oocytes.