Actin and tubulin synthesis in murine blastocyst outgrowths

The actin and tubulin contents of blastocysts grown in vitro for 72 h were estimated by densitometric analysis of Coomasie-blue stained SDS-gels. Actin represents 7% and tubulin 13% of total blastocyst outgrowth protein. The relative synthesis of these two proteins was measured by two-dimensional electrophoresis utilizing unlabeled and isotopically labeled actin and tubulin internal markers. Actin synthesis constituted 6.3% and tubulin synthesis 1.5% of the total protein synthesis. These values are not significantly different from those we have reported previously for mouse preimplantation blastocysts recovered from uteri. It appears then that the relative proportion of synthesis does not change significantly during the developmental period that encompasses the blastocyst stage and early implantation as represented by the in vitro hatching, attachment and outgrowth of the blastocyst. Data on the characteristics of growth and culture of the outgrowths is also presented.     

Protein metabolism in preimplanted mouse ova

Intracellular specific radioactivity of an amino acid (leucine) was measured in preimplantation mouse ova. The measured specific activity along with leucine incorporation rates allow calculation of the protein synthetic rate in mouse ova. Unfertilized ova, fertilized ova, two-cell ova, and blastocysts convert amino acid to protein at the rate of 8.60, 7.36, 6.92, and 56.68 × 10^?13 moles of amino acid per hour per ovum, respectively. The specific activity measurements also allowed the calculation of intracellular leucine pool size. For the unfertilized, fertilized, two-cell, and blastocyst stage, the endogenous leucine pool was 11.9, 15.9, 6.4, and 62.4 × 10^?14 moles of leucine, respectively. Most of the increase in pool size in the blastocyst probably results from the increase in total volume of cells. Protein degradation measurements indicated a marked difference between protein turnover in the two-cell and blastocyst stage. Approximately 10% of the protein at the two-cell stage turns over with a half-life of 18.3 hr and 35% of the protein at the blastocyst stage turns over with a half-life of 11.2 hr. The large remaining fraction of protein turns over much more slowly.     

Amino acid uptake and protein synthesis in preimplanatation mouse embryos

Amino acid uptake and cycloheximide inhibitable incorporation into protein are demonstrable in follicular ova, unfertilized eggs, and in mouse embryos ranging from the 1-cell to the late blastocyst stages. The rates of uptake and incorporation are low and relatively constant until the early blastocyst (day 3) stage of development when they increase 3- to 9-fold. The rate of uptake continues to increase during the fourth day (late blastocyst stage) of development, but, despite embryonic growth, incorporation into protein remains constant. By exposing embryos to leucine and lysine at different concentrations, it is possible to dissociate the processes of uptake and incorporation into protein from one another and to use the latter as a measure of protein synthesis. Taking the number of embryonic cells into account, it is postulated that the period between 8- to 16-cell stage (day 2) and the early blastocyst stage is the only one in which the synthesis of major amounts of protein based on new messenger RNA synthesis is occurring.Leucine and lysine uptake take place by a facilitated process, although lysine transport is not readily saturated. Inhibitors of energy metabolism do not significantly affect amino acid uptake, but they do inhibit protein synthesis. However, since the rate of transport is highly temperature sensitive (Q₁₀ ? 3) and leucine is accumulated against a concentration gradient, active amino acid transport appears to be present.     

Qualitative and quantitative changes in protein synthesis occur at the 8-16-cell stage of embryogenesis in the cow

Cow oocytes and preimplantation embryos were cultured in medium containing radiolabelled methionine and the proteins synthesized were analysed by one-dimensional electrophoresis and fluorography. Marked changes in the pattern of synthesis were observed at the 8-16-cell stage of development. Quantitatively, a gradual decrease in the rate of protein synthesis occurred between the zygote and 8-cell stage and then the rate increased progressively to the blastocyst stage. Incorporation of radiolabelled uridine into RNA was first detected at the 16-cell stage. Taken together, these results suggest that protein synthesis is programmed by maternal mRNA up to the 8-cell stage but switches to mRNA derived from the zygote genome between the 8- and 16-cell stage.     

Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.     

Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium

At least 71% of CF1 x B6SJLF1/J embryos developed from the 1-cell stage to the blastocyst stage in an optimum glutamine concentration of 1 mM, as long as glucose was present after the first 48 h of culture. Blastocysts raised under these conditions had significantly more cells than did blastocysts raised in CZB medium alone (glutamine present, glucose absent). Embryos raised in vivo accumulated 170-200 fmol glutamine/embryo/h at the unfertilized egg and 1-cell stages with a decline to 145 fmol/embryo/h at the 2-cell stage, followed by sharp increases to 400 and 850 fmol/embryo/h at the 8-cell and blastocyst stages. The presence or absence of glucose in the labelling medium had no effect on glutamine uptake by these embryos. Embryos raised in vitro accumulated 2-3 times more glutamine at stages comparable to those of embryos raised in vivo. In all cases in which 1-cell to blastocyst development in vitro was successful, glucose was present in the culture medium and the incremental uptake of glutamine between the 8-cell stage and the blastocyst stage was approximately 2-fold. This was also the increment for in-vivo raised embryos. When glucose was not present after the first 48 h, the 8-cell to blastocyst glutamine increment was not significant, and development into blastocysts was reduced. The results also show that glutamine can be used as an energy source for the generation of CO2 through the TCA cycle by all stages of preimplantation mouse development, whether raised in vivo or in vitro from the 1-cell stage. Two-cell embryos raised in vivo converted as much as 70% of the glutamine uptake into CO2, consistent with an important role for glutamine in the very earliest stages of preimplantation development. Cultured blastocysts appeared to convert less glutamine and the presence of glucose in the culture medium seemed to inhibit this conversion.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

Program of early development in the mammal: changes in patterns and absolute rates of tubulin and total protein synthesis during oogenesis and early embryogenesis in the mouse.

The absolute rates of total protein synthesis and tubulin synthesis during oogenesis and early embryogenesis in the mouse have been determined by measuring specific activities of the endogenous methionine pool and rates of incorporation of [³⁵S]methionine into total protein and tubulin. The absolute rate of protein synthesis decreases from 43 to 33 pg/hr/oocyte during meiotic maturation, while the size of the endogenous methionine pool remains essentially unchanged at 65 fmole/oocyte (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978, Proc. Nat. Acad. Sci. USA,75, 4160). The one-cell mouse embryo synthesizes protein at a rate of 45 pg/hr/embryo, so that fertilization is accompanied by about a 40% increase in the absolute rate of total protein synthesis. The eight-cell compacted embryo synthesizes protein at the rate of 51 pg/hr/embryo. The size of the endogenous methionine pool increases dramatically during early embryogenesis, from 74 fmole in the unfertilized ovum to 137 and 222 fmole in the one-cell embryo and eight-cell compacted embryo, respectively. Tubulin is one of the major proteins synthesized by the mouse oocyte and embryo since the absolute rate of tubulin synthesis is, on the average, 1.3% that of total protein synthesis. The absolute rate of tubulin synthesis decreases from 0.61 to 0.36 pg/hr/oocyte during meiotic maturation and then increases to 0.60 pg/hr/embryo in the one-cell embryo and to 0.66 pg/hr/embryo in the eight-cell compacted embryo. During meiotic maturation and early embryogenesis the direction and magnitude of changes in the rate of tubulin synthesis closely parallel those of total protein synthesis. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of the absolute rate of synthesis of the β subunit to that of the α subunit is about 2.0 throughout meiotic maturation and early embryogenesis.High-resolution two-dimensional gel electrophoretic analysis of [³⁵S]methionine-labeled proteins reveals that many of the newly synthesized proteins that first appear during meiotic maturation of the oocyte continue to be synthesized in the one-cell embryo. Nearly all of the proteins synthesized in the one-cell embryo are also synthesized in the unfertilized ovum, although some changes in the pattern of protein synthesis are associated with fertilization. Therefore, the developmental program for early embryogenesis in the mouse appears to be activated during meiotic maturation of the oocyte. These results are compared with those obtained using oocytes and embryos from nonmammalian animal species.     

Variations in the amounts of RNA polymerase forms I, II and III during preimplantation development in the mouse.

DNA-dependent RNA polymerase has been measured at various stages of preimplantation development in mouse embryos. The total RNA polymerase activity per embryo increases rapidly from the 8-cell stage to the blastocyst stage. Studies with low α-amanitin concentrations, which inhibit form II RNA polymerase, and high α-amanitin concentrations, which inhibit both form II and III RNA polymerases indicate that the relative proportions of the three forms change significantly during preimplantation development. The changes which occur in the types and levels of RNA polymerase appear to parallel corresponding changes in the synthesis of the major classes of RNA.     

Immunohistochemical demonstration of prostaglandin E-2 in preimplantation mouse embryos

An antiserum to prostaglandin (PG) E-2 and indirect immunofluorescence were used to demonstrate immunohistochemically the presence of PGE-2 in preimplantation mouse embryos. Fluorescence was observed in the cytoplasm of unfertilized 1-cell embryos to the blastocyst stage. The strongest fluorescence was detected at the 8-cell and morula stages. In embryos cultured from the 2-cell stage on, the fluorescence was observed in the cytoplasm of 4-cell embryos to the blastocyst stage. No differences were observed in the intensity and the distribution of the fluorescence between embryos in vivo and those in vitro. However, when blastocysts were cultured in a medium containing 100 microM-indomethacin, the fluorescence was diminished markedly. We therefore suggest that preimplanted mouse embryos contain PGE-2 during their early developmental stages and that the embryos synthesize the PGE-2.     

Glucose metabolism by preimplantation pig embryos

Pig embryos were collected, 2-7 days after oestrus, in modified BMOC-2 containing glucose as the only energy source. Embryos were incubated individually in medium containing [5-(3)H]-, [1-(14)C]- or [6-(14)C]glucose. Total glucose metabolism, as measured by [5-(3)H]glucose use, increased steadily from the 1-cell to the 8-cell stage. Total glucose use increased (P less than 0.05) at the compacted morula stage and was highest (P less than 0.05) at the blastocyst stage. Production of 14CO2 from embryos metabolizing [1-(14)C]glucose increased steadily from the unfertilized ovum to the 8-cell stage. Metabolism of [1-(14)C]glucose increased at the compacted morula stage (P less than 0.05) and continued to increase (P less than 0.05) to the blastocyst stage. Metabolism of [6-(14)C]glucose increased steadily from the unfertilized ovum to the compacted morula stage. Metabolism of [6-(14)C]glucose was highest (P less than 0.05) for the blastocyst stage. Percentage pentose phosphate pathway activity of total glucose metabolism before the 4-cell stage was higher (greater than 5%) than that of 8-cell to blastocyst stage embryos (approximately 1%). When embryo metabolism was determined on a per cell basis for each isotope, the compacted morulae stage (16 cells) had a higher total glucose metabolism than all other embryo stages (P less than 0.05), while early blastocyst (32 cells) and blastocyst (64 cells) stage embryos metabolized more [5-(3)H]glucose than all stages except compacted morulae (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The Effect of α-Amanitin on Nucleic Acid Synthesis in Preimplantation Mouse Embryos

It has been hypothesized that multiple forms of RNA polymerase may play a role in the control of development and differentiation in eukaryotic organisms. For this to be true, three criteria must be met. First, multiple forms of RNA polymerase must be demonstrated. Second, the relative proportion of the enzyme forms must be shown to change with development or differentiation. And third, the types of RNA synthesized must correlate with the types of RNA polymerase present at each developmental stage. We have previously reported data satisfying the first two criteria for preimplantation mouse embryos. The present paper probes the third criterion in this differentiating system.
It was found that although the proportion of the RNA polymerase enzyme forms changes from the 8-cell to the blastocyst stage of development, the types of newly synthesized nucleic acids at each of these stages were similar. Furthermore, inhibition of rRNA, mRNA, and tRNA, by α-amanitin, was identical for 8-cell and blastocyst embryos. The only difference between these two stages was that DNA synthesis in blastocysts was more sensitive to inhibition by α-amanitin than DNA synthesis in 8-cell embryos. We conclude that the synthesis of different classes of RNA by preimplantation mouse embryos is not simply controlled by changes in the levels of the multiple forms of RNA polymerase.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.     

Fucosylated glycoconjugates in mouse preimplantation embryos

Preimplantation mouse embryos were metabolically labelled with 3H or 14C-fucose to investigate the synthesis of fucosylated macromolecules. Scintillation counting revealed that there was a progressive increase in both total fucose taken up by the embryo and incorporation of fucose into TCA-precipitable material as embryos developed from the 4-cell to the blastocyst stage. This was reflected in the increasing intensity of bands on autoradiographs of radioactive fucose labelled proteins separated on 10% SDS-PAGs between the 4-cell embryo (at which stage bands were first detectable) and the blastocyst. Minor qualitative changes in fucoproteins were detected at the time of compaction and additional bands appeared at the blastocyst stage. Preliminary analysis of fucolipids in 6- to 8-cell embryos indicated that an approximately equal amount of fucose was incorporated into lipid and protein. Autoradiographs of semi-thin sections of 3H-fucose-labelled embryos showed substantial amounts of radioactive material in the vicinity of the plasma membrane both adjacent to other cells and facing the zona pellucida. These data would support a predominant role for fucoconjugates in cell surface events in the preimplantation embryo from the 8-cell stage.