Spectrin synthesis in the preimplantation mouse embryo

The preimplantation mouse embryo expresses two polypeptides, Mr 240,000 and Mr 235,000, that are immunologically cross-reactive with antibody to the alpha and beta subunits of mouse brain spectrin. We investigated the synthesis of the spectrin subunits in the Triton-soluble and Triton-insoluble fractions of fertilized eggs, two-cell embryos, compacted morulae, and blastocysts labeled with L-[35S]methionine. Synthesis of embryonic spectrin began in the Triton-soluble fraction with significant levels of alpha-spectrin synthesis first detected in the morula stage and significant levels of beta-spectrin synthesis detected in the blastocyst stage. Incorporation of newly synthesized alpha- and beta-spectrin into the cytoskeletal fraction took place in the blastocyst when equal amounts of both subunits were assembled. Previous studies have shown Triton-insoluble spectrin to be concentrated in regions of cell-cell contact in the embryo (J. S. Sobel and M. A. Alliegro, 1985, J. Cell Biol. 100, 333-336). The temporal and spatial correlation between the assembly of newly synthesized spectrin and its concentration in regions of cell apposition is consistent with the hypothesis that cell contact may influence the assembly of embryonic spectrin.     

De novo pyrimidine nucleotide synthesis in the preimplantation mouse embryo

Utilizing phosphonacetyl- -aspartate (PALA), the transition state analog which specifically inhibits aspartate carbamyl transferase, we have shown that the preimplantation mouse embryo in culture has a functioning de novo pyridmidine biosynthetic pathway. This pathway accounts for some of the carbon dioxide fixation into nucleic acids previously described. Inhibition of de novo pyrimidine nucleotide synthesis during 2-cell to 8-cell development does not prevent morula development, but does prevent blastocyst development in nearly all embryos. Inhibition of the morula to blastocyst transition is most likely caused by a diminished pyrimidine nucleotide pool. Both de novo and salvage pathways appear active from the 2-cell embryo through blastocyst formation.     

Effects of cordycepin on macromolecular synthesis and development in the preimplantation mouse embryo

Investigations were conducted to test the effects of cordycepin, a naturally-occurring analog of adenosine, on gene activity in preimplantation mouse embryos. Embryos were explanted into culture at the 2-cell, morula and blastocyst stages, and incubated in the absence or presence of cordycepin (5–100 μg/ml) to determine the effects of the drug on continued development and macromolecular synthesis. Cordycepin at concentrations exceeding 10 μg/ml caused a dose-responsive inhibition of cleavage and blastulation of embryos in culture. Exposure of morulae and blastocysts to cordycepin concentrations of 10–100 μg/ml produced a dose- and time-dependent suppression of RNA synthesis as measured by incorporation of [³H]uridine. Suppression in blastocyst-stage embryos was enhanced by preincubation, and reached 70% after 4 h at 100 μg/ml. Cordycepin (50–100 μg/ml) reduced synthesis of major RNA components detected by electrophoresis, blocked incorporation of radioactivity into fractions bound by olido(dT)-cellulose, and produced a time- and dose-dependent reduction of protein synthesis in blastocysts, causing a maximum inhibition of 25% after 4 h of preincubation at 50 μg/ml.     

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.     

Mitochondrial DNA in the mouse preimplantation embryo

Total DNA was extracted from mouse embryos that were collected from CD-1 random-bred females on Day 1 of pregnancy and cultured for up to 4 days in vitro, or from the reproductive tracts of pregnant females on Days 1, 3, 4 and 5 of pregnancy. Southern blot analyses with a cloned mouse mitochondrial DNA probe were performed to determine the relative levels of mitochondrial DNA in the zygote, morula, blastocyst and early egg cylinder stage embryos. The results indicated that the total amount of mitochondrial DNA does not change during development of the mouse embryo up to the egg cylinder stage and is not altered during in-vitro culture of the fertilized one-cell embryo to the blastocyst stage.     

Oligo(A) and double-stranded segments in polyadenylated and non-polyadenylated RNA from cytoplasm and nuclei of chick embryo

Chick embryonic RNA was fractionated by affinity chromatography on oligo(dT)-cellulose and poly(U)-Sepharose into three classes: poly(A)+RNA containing poly(A) segments of 100 and more residues, poly(A)-oligo(A)+RNA containing oligo(A) segments of about 25 residues, and poly(A)-oligo(A)-RNA which bound to neither of the beds used and which contained double-stranded segments of 300 and more base pairs. These three classes of RNA were found in cytoplasmic as well as in heterogeneous nuclear RNA. Double-stranded segments in hnRNA, unlike those in cytoplasmic RNA, were intermolecular in nature; this may explain the occurrence of "giant" molecules in hnRNA.     

Phosphofructokinase activity in the preimplantation mouse embryo

Summary Phosphofructokinase activity remains relatively constant during the preimplantation period in the mouse, with a low point at day 4 (approximately 3.0×1^–11 moles of substrate converted per embryo per hour).     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

Analysis of compaction in the preimplantation mouse embryo

An SEM analysis of the effects of tunicamycin, cytochalasin B, and colcemid has yielded insights into the process of compaction in the early mouse embryo. All three reagents block or reverse compaction and decrease the number of microvilli (MV), although some MV polarization is permitted. In addition, tunicamycin is shown to lessen cell adhesion even in compacted embryos. Cytochalasin B causes the formation of MV clumps some of which are preferentially localized to the apex or lateral ring region. Colcemid reverses compaction and, coupled with Pronase treatment, completely blocks compaction of uncompacted 8-cell embryos. Observations also suggest that MV polarization can occur only once but compaction (the close adherance and flattening of blastomeres) can be reversed and reinduced. Evidence is consistent with a three-step compaction process involving (1) cell surface recognition and attachment of a ring of lateral microvilli to adjacent blastomeres, (2) subsequent microfilament shortening in these lateral MV, and (3) maintenance of the compacted and polarized state by microtubules.     

Cytokeratin filament assembly in the preimplantation mouse embryo

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse 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.