Comparison of overall rates of RNA synthesis in implanting and delayed implanting mouse blastocysts in vitro

Implanting and delayed implanting mouse embryos were incubatedin vitro with [³H]uridine for 2–24 hr. The size and specific activity of the [³H]UTP pools were determined by means of a double isotope technique using copolymer synthesis with the [³H]UTP in the embryos, exogenous [¹⁴C]ATP, andE. coli RNA polymerase. Using the rate of incorporation of [³H]uridine into acid-insoluble material and the specific activity of the [³H]UTP pools, it was possible to calculate the overall rate of incorporation of uridine into RNA by the embryos. In implanting embryos it was constant for 24 hr. In contrast, the initial rate of uridine incorporation by the delayed implanting embryos was only 31% of that in implanting embryos (i.e., per cell); this increased steadily during the incubation period, reaching 81% of the rate in implanting embryos after 24 hr. This activation of RNA synthesis by delayed implanting embryosin vitro occurred in the absence of any uterine stimulatory factors. Further, it was shown that although 10% mouse serum would support trophoblastic outgrowthin vitro, it did not influence uptake, distribution of label into nucleotides, or rate of uridine incorporation into RNA in either implanting or delayed implanting embryos. Therefore, it is suggested that if depression and activation of metabolic activity in blastocysts are part of the mechanims of delayed implantation, and if trophoblast outgrowthin vitro is analogous to the process of implantationin vivo, then these two aspects of embryo activation are under different controls.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Expression of specific genes in early mouse embryos blocked by cytochalasin

Summary Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F₁-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.     

Effects of alpha-amanitin on RNA synthesis by mouse embryos in culture

Investigations were conducted to test the effects of alpha-amanitin on RNA synthesis in preimplantation mouse embryos. Exposure of embryos in culture to 1-100 microgram/ml alpha-amanitin produced a dose- and time-dependence suppression of total RNA synthesis as measured by incorporation of [3H]uridine. Synthesis of polyadenylated RNA in blastocyst-stage embryos was abolished by alpha-amanitin-treatment at concentrations and exposure times that suppressed total RNA synthesis by less than 15%. DNA-dependent RNA polymerase activity was measured in lysates of embryos at several stages of preimplantation development. alpha-Amanitin suppressed total polymerase activity assayed under ionic conditions favorable to the detection of RNA polymerase II. Electrophoretic analyses revealed that preincubation of blastocysts in 100 microgram/ml alpha-amanitin reduced labelling of cytoplasmic 28S and 18S RNA by inhibition of both synthesis and maturation of nucleolar 45SrRNA-precursor. This action of alpha-amanitin on nucleolar RNA synthesis cannot be correlated with the minimal suppression of nucleolar RNA polymerase activity and suggests that the synthesis and processing of rRNA may be under control of nucleoplasmic gene products.     

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.     

Macromolecular synthesis during plant embryogeny: rates of RNA synthesis in Phaseolus coccineus embryos and suspensors

A comparative study of RNA metabolism as an indicator of major changes of tissue organization, cell number, and physiology in the two developmentally and cytologically distinct parts of the bean embryo, the organogenetic part and the suspensor, was carried out. The metabolism of RNA was determined separately for these two parts of embryos removed aseptically from seeds at different times during embryogeny and incubated in culture medium containing ³H-adenosine. Equilibration of ATP in the nucleotide pool, ATP pool size and specific activity, total RNA content, rate of RNA synthesis in culture, rate of RNA synthesis and specific activity during embryogeny, and total protein content were determined. Synthetic activity of the suspensor was highest early in development and then declined, whereas synthetic activity of the organogenetic part increased throughout development. These changes may reflect developmental and functional differences in the two parts of the embryo.     

Effects of ultrasound on DNA and RNA synthesis in preimplantation mouse embryos.

Ultrasound is used extensively to monitor the growth of ovarian follicles in in vitro fertilization and embryo transfer (IVF-ET) programs, as well as to follow the progress of early pregnancy. There have been scattered reports in the literature that exposure to ultrasound may have an adverse effect on reproduction in the rat (Bologne et al: CR Soc Biol 177:381-387, 1983; Demoulin et al: Ann NY Acad Sci 442:146-152, 1985), and also in humans (Demoulin et al: Ann NY Acad Sci 442: 146-152, 1985). We report here that diagnostic levels of pulsed ultrasound did not affect either the number of embryos produced, or the ability to incorporate labelled precursors into DNA and RNA, respectively. Measurements of temperature elevation of ovaries exposed to ultrasound showed that neither controls nor experimental tissue exhibited temperature elevation greater than 1 degree C.     

RNA synthesis in nuclei isolated from early embryos of Xenopus laevis.

1. Rates of RNA synthesis in isolated Xenopus embryo nuclei decrease from blastula through gastrula and neurula stages to hatching tadpoles. 2. In blastula and gastrula nuclei, net synthesis of RNA continues for over 30 min, both in the presence of KCl at 0.4 M and in its absence. In nuclei from later stages, net synthesis continues for only about 10 min in the absence of KCl. 3. At low ionic strength, RNA synthesis in all nuclei is greater with optimum Mg-2+ (6 mM) than with optimum Mn-2+ (1 mM). At high ionic strength the reverse is true. 4. An unusual feature, which gradually disappears as development proceeds, is that curves relating RNA synthesis to KCl concentration show a peak at 0.1 M KCl. In blastula nuclei, RNA synthesis is more rapid at 0.1 M KCl than at 0.4 M. 5. This peak at low ionic strength is not observed in the presence of the initiation inhibitor rifamycin AF/013. It is concluded that the peak arises from initiation of RNA synthesis by an excess of RNA polymerases bound non-specifically to the isolated nuclei. The residual synthesis, representing elongation of chains that were initiated in vivo, still declines as development progresses. 6. In blastula nuclei, over half of the RNA synthesis is effected by polymerase II (inhibited by alpha-amanitin), the proportion remaining roughly constant with increasing ionic strength. In neurula nuclei, the proportion rises from about one-half to three-quarters. The initiation-dependent peak in blastula and gastrula nuclei is contributed by both alpha-amanitin-sensitive and alpha-amanitin-resistant enzymes.     

Poly(A) length,cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol.89, 362–378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [³H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 × 10⁷; one-cell embryo, 2.4 × 10⁷; late two-cell, 0.7 × 10⁷; late eight-cell, 1.3 × 10⁷; and early blastocyst, 3.4 × 10⁷. These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [³H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

RNA synthesis during early embryogenesis of mass cultured highly synchronized coleopteran embryos

Summary Bruchidius embryos are shown to be well suited for biochemical studies during early embryogenesis. Mass cultivation is easy, and highly synchronized embryos can be obtained in large numbers (10⁴–10⁵ eggs). A method for in vivo incubation is described which allows the labelling of newly synthesized RNA. The kinetics of³H-ruidine uptake, phosphorylation and incorporation into RNA are presented. By autoradiography, the distribution of newly synthesized RNA is shown. Thereby, stage-specific differences were found in the labelling pattern of vitellophage nuclei, of blastoderm nuclei and of the nuclei of pole cells. The labelling of the cytoplasm remains weak until cellular blastoderm is formed. During late blastoderm and at gastrulation this label increases markedly. Gel electrophoresis of isolated RNA shows that at cellular blastoderm formation most of the label occurs in a region between 18 S and 7 S. Later on, at the onset of gastrulation, the³H-uridine incorporation found in isolated RNA is raised about 10 fold and rRNA synthesis becomes prominent. In a chase experiment, the processing of precursor RNA molecules into shorter RNA species, especially into mature rRNA and 5S RNA, is shown. The advantages of theBruchidius embryo for the biochemical analysis of early RNA synthesis and the regulation of rRNA synthesis in insect embryos are discussed.Dedicated to Professor Dr. Dr. h. c. Bernhard Rensch at the occasion of his 80th birthday