Oogenesis Of Xenopus Laevis Research Articles

Poly(A) + RNA metabolism during oogenesis in Xenopus laevis

In this study, we have measured the synthesis and turnover of oligo(dT)cellulose-bound RNA [poly(A) + RNA] in Xenopus laevis oocytes at the maximal lampbrush chromosome stage (stage 3) and at the completion of oocyte growth (stage 6). Oocytes at both stages are shown to be active in the synthesis of poly(A) + RNA. In stage 6 oocytes, the mean rate of synthesis of stable poly(A) + RNA is 15% the instantaneous rate of synthesis, while the mean half-life of the unstable component is 1.6 hr. In contrast, the instantaneous rate of synthesis in stage 3 oocytes is about one-third that seen in stage 6, and most of it is devoted to the production of unstable species with an average half-life of 5 hr. Studies on the nuclear versus the cytoplasmic distribution of the newly synthesized poly(A) + RNA demonstrated that by the end of a 12-hr labeling period for stage 3 oocytes and a 24-hr labeling period for stage 6 oocytes, approximately half of the material was cytoplasmic. This cytoplasmic material had the same electrophoretic mobility as bulk poly(A) + RNA. Similarly, as with bulk poly(A) + RNA, little, if any, of the newly synthesized material was found to be polysomal. Also, poly(A) labeling studies indicated that the newly synthesized poly(A) + RNA was associated with the synthesis of poly(A) of the same length as that appearing on bulk poly(A) + RNA. Studies on the content of bulk oligo(dT)cellulose-bound RNA indicated that about 86 ng is present in both stage 3 and stage 6 oocytes. The continual synthesis of poly(A) + RNA throughout oogenesis in the absence of its accumulation led to the conclusion that it must be turning over. These data are discussed in relation to the hypothesis that bulk levels of poly(A) + RNA are maintained by continually changing rates of synthesis and degradation.

Quantitative determination of amplified rDNA and its distribution during oogenesis in Xenopus laevis.

The number of extra-chromosomal nucleoli and their rDNA content were determined during oogenesis in Xenopus laevis. The highly variable number of nucleoli (500 to 2,500) in oocytes of the same stage and from the same female or of different stages or from different females is not a measure of the extent of amplification. In all oocytes examined, a inversely proportional relation was found between the number of nucleoli in an oocyte and their mean rDNA content. These results indicate that there is no variation of the rDNA content of oocytes during oogenesis nor between oocytes of different females. The varying nucleolar numbers found in oocytes result thus from fusion and fission of pre-existing nucleoli. The determination of the rDNA content, in absolute units (35 pg), after amplification which occurs at the beginning of oogenesis, makes it possible to calculate the rDNA content of one nucleolus. This ranged from 0.7.10(-2) pg to 15.10(-2) pg, corresponding to about 500--11,000 cistrons of rDNA. No distinct size classes between these two extremes were observed.

Oogenesis in Xenopus laevis (Daudin). VI. The route of injected tracer transport in the follicle and developing oocyte.

In Xenopus laevis, vitellogenin (the yolk precursor) is synthesized in the liver and transported via the circulatory system to the ovary. In order to reach developing oocytes where it is sequestered, it must exit the circulatory system and traverse several follicular tissue layers including the theca, the follicle cell layer, and the vitelline envelope. This study demonstrates this pathway by means of electron-opaque tracers, and follows the fate of heterologous macromolecules after their incorporation into the ooplasm. The tracers used were horseradish peroxidase, iron dextran, ferritin, and thorotrast. The bulk of the tracers exit the circulatory system through gaps between adjacent capillary endothelial cells and migrate into the connective tissue theca, where they appear randomly dispersed. All tracers, except thorotrast, penetrate the basement membrane on the distal surface of the follicle cells and pass through channels between adjacent follicle cells into the vitelline envelope and to the surface of developing oocytes, where they are incorporated by endocytosis. Endosomes which contain tracer, and also presumably vitellogenin, fuse to form primordial yolk platelets. During this fusion process an extensive network of smooth-surfaced tubules arises in the peripheral ooplasm. Endosomes and/or primordial yolk platelets continue to fuse with each other, resulting in the growth of primordial platelets which move deeper into the ooplasm, where they are transformed into yolk platelets with crystalline main bodies. Peroxidase and iron dextran remain in the superficial layer of the platelet, while ferritin is present in both the superficial layer and the crystalline main body.

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A) S and poly(A) L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A) L and poly(A) S are associated with RNA which is heterogeneous in size. The two classes of poly(A) + RNA can be separated by affinity chromatography: Only poly(A) L + RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A) S + RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A) + RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A) L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A) S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A) + RNA molecules per oocyte increases throughout oogenesis from 2 × 10 10/previtellogenic oocyte [80–90% poly(A) L] to 20 × 10 10/full-grown oocyte (25–40% poly(A) L). It is argued that poly(A) S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A) + mRNA.

The synthesis and storage of histones during the oogenesis of Xenopus laevis

Further data, including two-dimensional gel electrophoresis and peptide mapping of newly synthesized proteins, confirms the view that oocytes make several types of histone. The newly synthesized histone is present in both nucleus and cytoplasm, but at a higher concentration in the oocyte nucleus and in great excess over the DNA binding sites. The unfertilized egg seems to contain a pool of histones detectable on two-dimensional electrophoretograms. The peptide maps of these proteins are consistent with their identification as histones. The egg contains enough histone to support nuclear replication through most of cleavage.

Accumulation of mitochondrial DNA during oogenesis in Xenopus laevis

The mitochondrial DNA (mtDNA) content of Xenopus laevis oocytes at various stages of oogenesis has been determined by molecular hybridization with 3H-labeled complementary RNA (cRNA). The previtellogenic oocyte less than 250 μm in diameter (stage 1) contains 0.95 ± 0.47 ng of mtDNA. Accumulation of mtDNA proceeds until stage 4 (500–750 μm diameter oocyte), by which time a steady-state level of 4.28 ± 0.40 ng/oocyte is attained. Using the hybridization assay, the stage 6 (full-grown) Xenopus oocyte contains 4.51 ± 0.69 ng of mtDNA, compared to the previously reported value of 3.8 ng determined by direct measurement on the unfertilized egg. There appears to be a reasonable correlation, therefore, between the termination of mtDNA accumulation and the dispersal of the juxtanuclear, mitochondrial aggregate (Balbiani body) at the onset of vitellogenesis in Xenopus. It is concluded that the enormous complement of oocyte mitochondria is accumulated well before the end of oocyte growth and is maintained at a constant level during the remainder of oogenesis, through maturation, fertilization, and on into early development.

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.

Oogenesis in Xenopus laevis (Daudin). III. Localization of negative charges on the surface of developing oocytes

Developing oocytes and coelomic eggs of mature HCG-stimulated Xenopus females were dissected from the ovary, fixed in glutaraldehyde, and treated with a positively charged solution of colloidal iron. Electron micrographs reveal anionic sites preferentially localized in or on the plasmalemma covering the distal portions of the numerous microvilli of Stage IV oocytes. Both prior to and subsequent to Stage IV (the peak of vitellogenic activity), the anionic sites on the oolemma are more randomly distributed. No iron particles appear in endocytotic pits of the oolemma at any stage of development, but endocytotic pits of follicle cells are labeled. There are few if any anionic sites in the vitelline envelope. The assumption that the negative charges are due to sialic acid moieties of surface mucopolysaccharides was borne out by treatment with neuraminidase. Oocytes subjected to the enzyme and subsequently fixed and treated with colloidal iron show a marked reduction in the amount of labeling. These results are discussed in the light of earlier evidence regarding the endocytosis of vitellogenin, a negatively charged molecule.

Oogenesis in Xenopus laevis (Daudin). IV. Effects of gonadotropin, estrogen and starvation on endocytosis in developing oocytes.

This study was designed to explore the relationship of estrogen, human chorionic gonadotropin (HCG), and food availability to endocytosis in developing oocytes. When estrogen alone is administered to an animal, large amounts of vitellogenin are synthesized by the liver and secreted into the circulatory system, where it accumulates. Under these conditions there is no evidence of endocytosis at the surface of the oocytes. Other studies have shown that following HCG injection into estrogen-treated animals, vitellogenin is removed from the circulation and the oocyte surface is highly contoured and displays endocytotic activity. Food deprivation has much the same effect on oocyte endocytosis as does estrogen. When animals are given HCG and subsequently starved for 20 days, developing oocytes show little endocytotic activity. We conclude that HCG acts to promote or stimulate endocytosis in developing oocytes while estrogen and/or starvation inhibits this process.

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.

Oogenesis in Xenopus laevis (Daudin) : II. The induction and subcellular localization of tyrosinase activity in developing oocytes

Tyrosinase activity is increased at specific stages of development in Xenopus laevis oocytes in mature females by an injection of 1000 units of human chorionic gonadtropin (HCG). Enzyme activity is stimulated slightly in stage II oocytes, greatly (5- to 6-fold) in stage III and early stage IV oocytes, slightly in late stage IV, and not at all in stage V oocytes. Tyrosinase activity has been localized cytochemically in oocytes by the DOPA-reaction. The DOPA-reaction product is found in the distal cisterna of the Golgi complex and in an anastomosing network of smooth-surfaced tubules associated with the Golgi complex. No reaction product is found in the clustered elements of smooth endoplasmic reticulum which gives rise to the premelanosomes. Substantial melanization of the premelanosomes does not occur until the DOPA-positive Golgi complexes move into proximity with the premelanosomes at the oocyte periphery. Biochemical assay of the isolated melanin granules shows that premelanosomes isolated from stage III and IV oocytes contain significant tyrosinase activity. This activity appears to decrease in the later stages of melanization. It is concluded that the metabolic activities leading to the formation of oocyte pigmentation are stimulated by gonadotropins and the degree of response to the stimulation is quantitatively regulated according to parameters typical of the specific stage of oocyte development.

The synthesis of amplified ribosomal DNA.

The DNA which codes for the 28 and 18 S ribosomal RNA molecules (rDNA) has been shown to undergo a 1000-fold amplification during the early stages of oogenesis in Xenopus laevis (Brown and Dawid, 1968; Perkowska et al., 1968; Gall, 1968). The chromosomal rDNA, which includes about 450 copies of the ribosomal gene at each nucleolar organizer (Brown and Dawid, 1968), and the amplified rDNA, organized in circles with different numbers of repeats in each circle (Miller, 1965), are identical for the lack of methylated bases in the amplified sequences (Dawid et al., 1970; Wensink and Brown, 1971). About 50% of the basic sequence of these tandem repeats is not contained in the mature rRNA (Brown and Weber, 1968; Birnstiel et al., 1968).

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10 6 daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.

Evolution of the nucleoli during oogenesis in Xenopus laevis studied by electron microscopy.

ABSTRACT Xenopus laevis tadpoles and toads were killed at several ages. The structure of the nuclei of the germinal cells has been observed by light and electron microscopes. We distinguish 11 successive stages in nucleolar structure: (1) a single, essentially granular nucleolus in the oogonium (10 μm diameter), (2) a reticulated nucleolus in the leptotene oocyte, (3) fragmentation of this nucleolus into a few smaller nucleoli, (4) multiple tiny nucleoli appearing in the cap of the pachytene oocyte, (5) enrichment in the fibrillar constituent of these intra-cap nucleoli, (6) grouped spherical nucleoli, with well segregated granular and fibrillar constituents, as the disintegration of the cap is going on (diplotene A oocyte, 30 μm diameter), (7) dispersion of those nucleoli in the nuclear sap (diplotene B oocyte, 50 μm diameter), (8) formation of long, ribboned nucleoli with multiple DNA-rich spots (diplotene C oocyte, 100 μm diameter), (9) fragmentation of the nucleolar ribbons into multiple spherical nucleoli with excentric fibrillar core and granular cortex (diplotene D oocyte, 150 μm diameter), (10) multiple purely fibrillar nucleoli (diplotene E oocyte, between 150 and 400 μm diameter), and (11) multiple classical nucleoli with concentric fibrillar core and granular cortex (diplotene F oocyte, between 400 and 1000 μm diameter). The multiplication of the nucleoli in Xenopus laevis may occur successively (a) by the fragmentation of the single oogonium nucleolus at the leptotene stage, (b) by de novo formation of nucleolar bodies inside the cap at the pachytene stage, and (c) by the growth of those nucleoli lying free in the nucleolar sap at the early diplotene stage. They evolve into nucleolar ribbons which later on fragment into spherical bodies. Four successive phases during the growth of an oocyte can be distinguished with respect to the ribosomal system. (I) The first phase is characterized by the nucleolar DNA amplification. (II) During the second phase, the multiplication of the nucleoli is going on. Ribosomes are present in the cytoplasm and the rate of cellular growth is very high. (III) During the third phase, the synthesis of rRNAs seem to be repressed while the synthesis of heterogenous small RNAs is going on. Ribosomes are no longer visible in the cytoplasm. The nucleoli are purely fibrillar. The rate of cell growth is lower than in the preceding phase. (IV) During the fourth (= Duryee lampbrush stages 3—6), or vitellogenic phase, rRNAs are actively synthesized and numerous ribosomes appear in the cytoplasm. The nucleoli have the classical structure and the rate of growth is about the same as during phase III.

RNA-dependent DNA polymerase: possible role in the amplification of ribosomal DNA in Xenopus oocytes.

2',5' - dimethyl - N(4')benzyl - N(4') - [desmethyl]rifampicin, a derivative of rifampicin, is used to acquire autoradiographic evidence that RNA-dependent DNA synthesis is involved in gene amplification during the early oogenesis of Xenopus laevis.

RNA synthesis in early oogenesis of Xenopus laevis

RNA synthesis in early oogenesis of Xenopus laevis