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R Bachvarova

Publications and source records attributed to R Bachvarova.

At least 19 recordsLinked to original sources
PubMed1997

Early chick embryos in vitro.

In 1955, Denis New described a technique for the in vitro culture of early avian embryos that has formed the basis for nearly all of the experimental embryological studies performed on these species since that day. Many modifications to this technique have also been described in these four decades for specific experimental purposes. Here, we review the effects of some parameters that appear to be important for different aspects of the growth of embryos in this type of culture, and conduct a small experimental comparison between different modifications of the technique as described by various authors. We conclude that the original technique still compares favorably with its alternatives.

Animals↗
PubMed1994

Polyadenylation and deadenylation of maternal mRNAs during oocyte growth and maturation in the mouse.

We previously showed that, during mouse oocyte maturation, specific maternal mRNAs (actins) are deadenylated, while others (hypoxanthine phosphoribosyltransferase:HPRT) are adenylated. As in other systems, these changes can be correlated with changes in translational activities. Maturation-specific polyadenylation in Xenopus depends on the presence of a U-rich cytoplasmic polyadenylation element (CPE) close to the 3' end of the RNA. RNAs that lack CPEs appear to be deadenylated by default when meiosis resumes. We show here that this default program also applies to maturing mouse oocytes. Microinjected beta- and gamma-actin 3' UTR (untranslated region) transcripts lacking CPEs but including polyA tails (100-200 N) behave as endogenous maternal actin mRNAs and are deadenylated by maturing oocytes. "Nonsense" transcripts that do not include CPEs, but that do contain polyA tails, are also deadenylated. beta- and gamma-Actin 3' UTRs with short polyA tails (50-80 N) are stable and exhibit no detectable change in adenylation when injected into growing, full-grown, or maturing oocytes. In contrast, HPRT 3' UTRs, which include the CPE UUUUAAAU and a short polyA tail (50 N), are polyadenylated during maturation. HPRT 3' UTR transcripts with long polyA tails (100-200 N) are more extensively deadenylated by growing and full-grown oocytes that retain germinal vesicles than by maturing oocytes. The presence of CPEs may be required for polyA tail shortening and translational inactivation of stable mRNAs during oocyte growth and subsequent selective readenylation and translation during meiotic maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1989

Amounts and modulation of actin mRNAs in mouse oocytes and embryos.

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.

Actins↗
PubMed1988

Small B2 RNAs in mouse oocytes, embryos, and somatic tissues.

RNAs of full-grown mouse oocytes, ovulated eggs, embryos, and somatic tissues have been analyzed on Northern blots for the presence of small transcripts homologous to the B2 element, a repetitive 180-nucleotide (N) sequence in the genome, using a single stranded RNA probe. In addition to the heterogeneous 200- to 600-N polyadenylated group reported by others, cytoplasmic RNA contains discrete nonadenylated species of B2-related RNA, approximately 100, 120, 155, and 180 N in length. During meiotic maturation of oocytes, the 200- to 600-N group declines and the 155-N species becomes more prominent. Upon hybridization to oligo(dT) and cleavage with RNase H to remove poly(A) regions, the 200- to 600-N group is removed, the 180-N species increased greatly, and the 155-N species increased slightly. Essentially all of the 200- to 600-N species bind to poly(U) sepharose. We conclude that polyadenylation rather than run-on transcription of B2 elements accounts for most of the heterogeneity of the 200- to 600-N group and that some deadenylation and cleavage take place during maturation. Small B2 RNAs make up 0.04% of total RNA in oocytes and eggs, 6- to 9-fold more than in brain. For comparison, a known small RNA, 4.5 S RNA, is relatively sparse in oocytes and almost absent in eggs. The 100-N B2-related species has been tentatively identified as 4.5 SI RNA; relative to total RNA, it remains approximately constant in oocytes and somatic tissues. During development to the blastocyst stage, small B2 RNAs per embryo increase severalfold, but decline as a fraction of total RNA. In postimplantation development, they continue to decline toward the level found in brain. Expression of B2 transcripts in hnRNA rises around 10 days of development to the level found in brain. The time course of expression of small B2 RNAs suggests an important role in development.

Age Factors↗
PubMed1988

Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse.

Previous work has shown that more than 50% or about 50 pg of polyadenylated RNA found in the full-grown mouse oocyte is deadenylated or degraded during meiotic maturation. Here we show that rRNA declines by 60 pg during this period, accounting for most of the 80-pg decline in total RNA and indicating that a significant amount of mRNA is deadenylated but not degraded during maturation. Actin mRNA is deadenylated at about 7 hr of in vitro maturation, following the decline in its translation. The poly(A) tail on hypoxanthine phosphoribosyltransferase (HPRT) mRNA is elongated at 7 hr of maturation, preceding an increase in HPRT activity. Actin mRNA is partially degraded in the one-cell embryo and falls to near the limit of detection in the late two-cell stage, while HPRT mRNA shows no change in early two-cell embryos, but is deadenylated and declines greatly during the two-cell stage. In aging unfertilized eggs, most of these changes occur on a delayed schedule. The various species of alpha-tubulin mRNA are largely deadenylated and more than half are degraded during maturation. Taken together with other published results, we conclude that each mRNA has its own pattern of changes in the length of the poly(A) tail (correlated with translation) and degradation during the period of maternal control of protein synthesis, and, for those examined, the maternal mRNAs remaining in the early two-cell embryo are degraded to low levels by the late two-cell stage.

Animals↗
PubMed1985

Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos.

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.

Animals↗
PubMed1985

Gene expression during oogenesis and oocyte development in mammals.

Mouse oocytes progress through early meiotic prophase during fetal life and reach the diplotene stage by birth. During prepubertal and reproductive life, oocytes are continuously selected to grow from the pool of small primordial oocytes. Growing oocytes reach full size in 2 weeks, and full-grown oocytes are present in rapidly enlarging follicles for about 5 days before meiotic maturation and ovulation. RNA synthesis during early meiotic prophase, as estimated from [3H]uridine incorporation followed by autoradiography and from electron microscopic analysis of nuclear components, proceeds at a moderate rate throughout except for a brief period in early pachytene when synthesis is low or absent. RNA synthesis continues in primordial oocytes at a moderate rate. Incorporation studies, electron microscopic analyses, and particularly measurements of ongoing RNA polymerase activity (completion of initiated chains as analysed in tissue sections) indicate a distinctly increased rate of synthesis during oocyte growth over that of primordial oocytes, followed by a decline in full-grown oocytes. During growth, this rate increases severalfold. The absolute rate of synthesis of heterogeneous nuclear RNA (using rRNA as a standard) during mid-growth is very rapid, but nevertheless still much lower than that in typical lampbrush chromosomes. Most of the hnRNA turns over with a half-life of about 20 min, as is typical in somatic cells. Newly synthesized mRNA-like RNA enters the cytoplasm at about one-half the rate of rRNA, and about one-third of the ribosomes and one-fourth of the mRNA appear in polysomes. In full-grown oocytes, the rate of synthesis falls distinctly, but a significant level of synthesis continues until it essentially ceases at breakdown of the germinal vesicle. During meiotic prophase, chromosomes are most compact at pachytene and unfold lateral projections as RNA synthesis increases in late pachytene-early diplotene. In primordial oocytes, the diplotene state of chromosomes is obvious in most mammals, but in rodents the chromosomes are more evenly dispersed and are said to be in a dictyate state, although they are still presumably in a diplotene configuration. The chromosome core, which is present in leptotene through early diplotene stages, apparently disappears in the dictyate stage.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Changes in total RNA, polyadenylated RNA, and actin mRNA during meiotic maturation of mouse oocytes.

The total RNA content of mouse oocytes, as measured by ethidium bromide fluorescence, was found to decrease by 19% during meiotic maturation (ovulated eggs contain 19% less RNA than full-grown oocytes). Consistent with these results, prelabeled stable RNA of full-grown oocytes decreased by about 20% during in vitro maturation. Polyadenylated RNA represented 19% of total prelabeled RNA in full-grown oocytes and 10% in oocytes matured in vitro, confirming previous results on in vivo prepared material. To distinguish between deadenylation and degradation for one mRNA, the amount and state of adenylation of actin mRNA was examined using Northern blots of oocyte RNA probed with a nick-translated beta-actin cloned chicken cDNA. The results showed that the amount of actin mRNA remained similar during maturation, but its molecular weight decreased slightly. Experiments in which RNA was treated with oligo(dT) and RNase H demonstrated that the actin mRNA was deadenylated during maturation, when actin synthesis is known to decline. These results indicate that the previously defined loss of bulk RNA and changes in the state of adenylation of mRNA during the first 11/2 days of embryogenesis actually begin during the 12 hr of meiotic maturation preceding fertilization.

Actins↗
PubMed1985

Repetitive sequence transcripts and U1 RNA in mouse oocytes and eggs.

Others have reported that about two-thirds of the polyadenylated RNA of sea urchin or frog eggs contains short interspersed repetitive sequence transcripts, a much larger proportion than that found in mRNA of somatic cells. Thus, it appears that incompletely processed transcripts accumulate in these oocytes. Also, in what may be a related phenomenon, the nuclear concentration of U1 RNA (involved in processing hnRNA) decreases during growth of frog oocytes. To pursue this question in mammals, Northern blots of RNA from mouse oocytes and eggs collected before and after meiotic maturation were probed with genomic clones containing rodent Alu-equivalent sequences. The Alu sequence is the predominant short interspersed repetitive element in the genome and is abundant in hnRNA. When compared on the basis of mRNA content, the oocyte and egg RNA contained less short repetitive sequence transcripts than liver or brain cytoplasmic RNA. Using a U1 RNA-specific probe, the concentration of U1 RNA in mouse oocyte nuclei was found to be quite similar to that in somatic cells, and U1 RNA was stable during meiotic maturation. These results suggest that processing of transcripts in mouse oocytes does not possess the unusual features observed in lower animals.

Animals↗
PubMed1983

Half-lives and relative amounts of stored and polysomal ribosomes and poly(A) + RNA in mouse oocytes.

Growing mouse oocytes were labeled in vitro with [3H]uridine and chased for 2 or for 7 days to estimate the relative amounts of RNA appearing in different fractions and to follow their turnover. Oocytes were lysed and thoroughly dispersed in the presence of 1% DOC, and centrifuged on sucrose gradients to separate polysomes from smaller components not engaged in translation. After the short chase, one-third of the labeled ribosomes appeared in EDTA-sensitive polysomes. The proportion of ribosomes in both fractions remained stable during the long chase, demonstrating no net flow from one fraction to the other. When gradient fractions were analyzed by poly(U) Sepharose chromatography, it was found that about 20% of the labeled poly(A)+ RNA appeared in polysomes after the short chase. The half-lives of stored and translated mRNA were followed relative to stable rRNA during the long chase. Stored mRNA was completely stable, but translated mRNA turned over with a t1/2 of about 6 days. Other methods for separating stored from translated components were not successful, including sedimentation of putative large complexes (fibrillar lattices) containing stored components, or chromatography of lysates on oligo(dT)-cellulose. Results presented here combined with our previous results demonstrate that, during meiotic maturation, the percent of labeled stable RNA which is polyadenylated declines from 19 to 10%, suggesting deadenylation or degradation of half of the accumulated maternal mRNA.

Animals↗
PubMed1982

rRNA accumulation and protein synthetic patterns in growing mouse oocytes.

The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.

Animals↗
PubMed1982

Morphology and transcriptional activity of mouse oocyte chromosomes.

Lampbrush chromosomes of growing amphibian oocytes carry thousands of lateral loops each of which consists of a chromatin fiber heavily encrusted with nascent ribonucleoprotein fibrils. These are believed to be responsible for the accumulation and maintenance of RNA transcripts found stored in the egg. In the case of mammalian oocytes, lampbrush chromosomes are most likely to occur during the major growth phase and also possibly during pachytene-early diplotene stages of meiosis. We have examined pachytene and early diplotene mouse oocyte chromosomes through the light microscope using sections of plastic-embedded material and air dried spreads stained with either silver nitrate or methyl green pyronin. Our results indicates that the projections radiating from the chromosomal axis are bundles of chromatin fibers rather than single fibers covered with an ribonucleoprotein matrix. These bundles may represent partially unfolded chromomeres. The axis itself could be partially dispersed revealing threads surrounding a fine linear element. -- Little is known about chromosome structure in growing mammalian oocytes, the stage when transcriptional activity is likely to be most rapid. In our preparations chromosomes at this stage appear as partially condensed fuzzy threads of relatively uniform width. In some cases, the fuzzy thread is seen to contain a dense linear core in the center. Thus, during the growth phase, the chromosomes retain a relatively condensed axis, a characteristic of meiotic chromosomes in general. RNA-containing material is found diffusely spread within the nucleus but not specifically associated with the chromosomes. Electron microscopic analysis of spread chromatin from growing oocytes demonstrates that most transcription units possess only one or two nascent ribonucleoprotein fibrils while a few have more. These and other published data indicate that mouse oocytes do not have true lampbrush chromosomes at any stage of their development.

Animals↗
PubMed1981

Mouse egg ribosomes: evidence for storage in lattices.

The ribosomes of mouse ova were labeled by exposure of growing ovarian oocytes to [3H]uridine. The ribosomes of ova lysed and dispersed in hypotonic medium were contained in particles with an unusually low buoyant density in CsCl density gradients. These particles sedimented at 9000g or less, and electron microscopy of the pellet revealed ribosome-like particles embedded in a fibrillar network. These results indicate that the ribosomes are present in a proteinaceous superstructure, probably the lattices seen in situ by transmission electron microscopy.

Animals↗
PubMed1980

Development of naked growing mouse oocytes in vitro.

Half grown oocytes were released from follicles of 7-day-old mouse ovaries during overnight culture. Naked oocytes free of attached follicular cells were cultured in MEM plus 10% fetal calf serum over the developing monolayer of ovarian cells. Oocytes increased in diameter for a period of at least a week at 1.6 micron per day, or 70% of the in vivo growth rate. The ultrastructure of cytoplasmic organelles in cultured growing oocytes was normal. During the second week, about 35% underwent spontaneous fragmentation, and at least 10-20% resumed meiotic maturation. We conclude that a significant fraction of naked oocytes reached a functional state comparable to that of normal full grown oocytes at a rate approaching the in vivo rate. Free oocytes with attached cells grew to a larger average diameter than naked oocytes, and incorporated 3H-leucine 75% more rapidly. Naked oocytes cultured in the absence of ovarian cells did not grow, and died when pyruvate was omitted from the medium. Naked oocytes did not grow when cultured over primary mouse fibroblasts, L-cells, CHO cells, or hepatoma cells. Naked oocytes separated from the monolayer of ovarian cells by a 0.7 mm layer of agar grew at 60% of the rate of control oocytes.

Animals↗