Elongation factor 1 alpha (EF-1 alpha) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis.

In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1 alpha (EF-1 alpha) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1 alpha is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1 alpha becomes evenly distributed in the cytoplasm. The main phase of EF-1 alpha accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).

Mitochondrial gene expression during Xenopus laevis development: a molecular study.

Mitochondrial gene expression has been analysed during embryonic development of Xenopus laevis; the relative amounts of 12S and 16S ribosomal RNAs and of most mitochondrial messenger RNAs were determined by slot-blot and Northern-blot hybridization experiments with specific mitochondrial DNA probes. The rRNA content per embryo remained constant during early development, confirming earlier results of Chase and Dawid (1972, Dev. Biol., 27, 504-518.); on the contrary, all mRNAs decreased abruptly after fertilization within a few hours (by a factor of 5-10), remained at a very low level up to the late neurula stages and increased again during organogenesis. Since the mitochondrial DNA content does not vary during this period molecular analyses as well as biological observations suggest that the mitochondrial genome is completely inactivated at the beginning of embryonic development. The amounts of rRNAs and mRNAs evolve therefore as a function of time only according to their half-lives. Mitochondrial RNA accumulation resumes subsequently with a high rate when the general transcription in the embryo is starting again, and this occurs before the resumption of DNA replication in the organelle. It appears that the Xenopus embryonic development represents a quite clear example of regulation of the mitochondrial expression at the level of transcription.

Changes in D-loop frequency and superhelicity among the mitochondrial DNA molecules in relation to organelle biogenesis in oocytes of Xenopus laevis.

Changes in mitochondrial DNA (mtDNA) replication activity are known to occur during oogenesis in Xenopus laevis. Electron microscopic and electrophoretic analyses carried out on mtDNA molecules at different vitellogenic stages show that 1. The frequency of displacement loop (D-loop) forms is correlated with the intensity of mitochondrial biogenesis. 2. Most of the native molecules as well as the D-loop carrying ones are superhelical. 3. Four families of different superhelicity may be distinguished and D-loops are found only in the most superhelical ones. To account for the changes in the frequencies of the D-loop forms and of the different topological types during cell differentiation, it is suggested that the initiation of a new replication occurs only on the most superhelical molecules and that some control of superhelicity may exist in mitochondria.

DNA topoisomerase I from mitochondria of Xenopus laevis oocytes.

Activity of DNA topoisomerase I has been characterized in extracts of mitochondria purified from Xenopus laevis oocytes. Several lines of evidence have been obtained for the intramitochondrial localization of the enzyme. The mitochondria-associated of DNA topoisomerase I represents 1% of the activity recovered from a total ovary population of oocytes. The enzymes has been purified by DEAE-cellulose, phosphocellulose and double-stranded DNA cellulose chromatography and its properties compared to those of its nuclear-cytosolic counterpart purified by the same purification steps. Both enzymes appear to possess very similar if not identical physico-chemical and catalytic properties. Neither enzyme shows DNA gyrase activity and they are both equally sensitive towards trypanocide drugs such as ethidium bromide and berenil. The mitochondrial enzyme is able to remove positive superhelical turns and possibly acts as a swivelase during replication of mitochondrial DNA. These results confirm the pioneering work of Fairfield et al. (1979, J Biol. Chem. 254, 9354) on the presence of a DNA topoisomerase I in mitochondria. However our results support the conclusion that in Xenopus laevis oocytes the mitochondrial and nuclear cytosolic DNA topoisomerase I are in all likelihood one and the same enzyme.

Development of the mitochondrial mass and accumulation of mtDNA in previtellogenic stages of Xenopus laevis oocytes.

The development of the mitochondrial mass of the previtellogenic oocytes of Xenopus laevis has been analysed by a morphometric method and it has been correlated with the growth of the occyte itself. The study which was performed with oocytes from females of very different ages shows that the relative size of the mitochondrial mass in the smallest oocytes decreases gradually with the age of the females. Cytophotometric analysis of the Feulgen-stained mitochondrial mass enabled the mtDNA content of that structure to be quantified during its development. This development involves about 12 rounds of replication out of the total 16--17 rounds of replication during complete differentiation of large oocytes. It is possible to estimate the mean replication rate of the mtDNA during the course of the previtellogenic period. The calculated rate of replication is very high in the mitochondrial mass of all the growing oocytes of very young females(about 1--2 months old), then it decreases rapidly when the oocytes reach 120 microns in diameter. These results suggest that the mitochondrial masses found in small oocytes of old females do not correspond to sites of active mitochondrial biogenesis but rather they represent a physiologically arrested collection of organelles. Thus the aggregation of mitochondria is the cytological expression of rapid and strictly localized mitochondriogenesis only in very young females.

KINETIC ANALYSIS OF ENTIRE OOGENESIS IN XENOPUS LAEVIS.

In our breeding conditions (without artificial hormonal stimulation) two years after metamorphosis are necessary for a Xenopus laevis female to produce mature oocytes. Four periods of different growth rates can be distinguished into this first wave of oogenesis: 1) oocytes reach 120 µm in diameter (early stage I according to Dumont (5)) in a few weeks after metamorphosis; 2) growth slows down and the size of 250-300 µm (late stage I) is obtained 6-7 months later; 3) a rapid growth resumes during vitellogenesis (stages II, III and IV) and a 1,000-1,100 µm diameter is reached in 2-3 months; 4) the last period spans for a year and the oocyte grows up to 1,200 µm. This phase covers many physiological changes and it should be critical to size carefully the oocytes for biochemical investigations and comparisons. At last most of the oocytes in a young female do not proceed through this entire oogenesis, they are stopped at the end of the second growth phase (about 250 µm in diameter).