The transition from maternal to zygotic control of development occurs during the 4-cell stage in the domestic pig, Sus scrofa: quantitative and qualitative aspects of protein synthesis

A study was conducted to identify the embryonic stage when the zygotic genome begins to direct development and to characterize protein synthesis in pig oocytes and embryos. Reproductive tracts of gilts were flushed to obtain unfertilized oocytes (UFO), zygotes (Z), 2-, 4-, and 8-cell embryos, compact morulae (M), initial blastocysts (IB), blastocysts (B), and hatched blastocysts (HB). Pig eggs and embryos were cultured in medium containing 1 microM L-[35S]methionine and evaluated for amino acid uptake, incorporation of the radiolabel into protein, and qualitative changes in protein profiles specific to each cleavage stage. Unfertilized oocytes sequestered 65.7 fmol methionine/4 h/embryo. Uptake of methionine decreased (p less than 0.05) from the Z (49.4), 2-cell (41.8), and 4-cell (37.6) embryonic stages to the M (8.97 fmol/4 h/embryo) stage. This downward trend was reversed at the IB, B, and HB stages when uptake increased to 37.3, 50.3, and 84.2 fmol/4 h/embryo, respectively. Incorporation of methionine into protein followed a similar pattern, being relatively higher in the UFO (21.0), Z (20.5), and 2-cell stages (16.0); decreased (p less than 0.05) at the 4-cell (6.67), 8-cell (6.84), and M (6.16) stages; and increased (p less than 0.05) at the IB (28.0), B (41.5), and HB (69.6 fmol/4 h/embryo) stages. Differences in protein profiles were observed for UFO, Z, 4-cell, and M stages using lysates of single embryos, one-dimensional SDS-PAGE, and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro

Preimplantation goat embryos were cultured with or without goat oviduct epithelial cells in Earle's 199 medium + 10% goat serum (E199 + 10%GS), and in three different simple chemically defined media. In-vivo development was characterized by an extended 8- to 16-cell stage followed by a rapid cleavage rate in the next 3 cell cycles. Culture of 1-8-cell embryos in Medium E199 + 10%GS led to cleavage arrest at the 8-16-cell stage, while in the chemically defined media embryos developed poorly and a high percentage failed to pass the 8-16-cell stage. In co-culture, however, a high percentage (77% and 96%) of 1-2-cell and 4-8-cell embryos, respectively, developed beyond the 16-cell stage. In co-culture, 1-2-cell embryos maintained cleavage rates equivalent to those in vivo until the 8-cell stage, but thereafter cell numbers lagged behind those in vivo, and by 168 h after ovulation, cell numbers (+/- s.e.) in vitro were 47.6 +/- 7.9 compared to 238 +/- 27.2 in vivo (t = 6.93, P less than 0.001). The results demonstrate that co-culture of embryos with oviduct cells allows a high percentage of embryos to develop through the period of cleavage arrest, providing a favourable environment for development through the 1-16-cell stages but a less adequate environment for development to the blastocyst stage.     

Nucleic acid synthesis in preimplantation mouse embryos

Summary Mouse embryos were collected at the 2-cell stage, cultured in vitro in the presence of³H deoxyuridine or uridine for 6 or 4 h and autoradiographed.Deoxyuridine is actively incorporated into the DNA of cleaving mouse embryos indicating the existence of thymidylate synthetase activity at least at the 4-cell stage and presumably already before this.RNAase treatment of embryos squashed on slides shows a weak but obvious incorporation of uridine into DNA of cleaving mouse embryos, from the 4-cell stage onwards; this incorporation is totally inhibited by hydroxyurea. The reduction of ribonucleotides to deoxyribonucleotides is a metabolic pathway already required for cleavage, as shown by hydroxyurea experiments.The second polar pody, known to incorporate thymidine, is unable to incorporate either deoxyuridine or uridine.     

Cyclin B1 levels in the porcine 4-cell stage embryo

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor alpha-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without alpha-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with alpha-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.     

DNA synthesis in the second and third cell cycles of mouse preimplantation development. A cytophotometric study

Female Swiss mice were sacrificed at 2 h intervals between 16–30 and 40–56 h after insemination. One-, 2- and 4-cell embryos were stained by the Feulgen method and cytophotometric measurement of their nuclear DNA content was carried out. The cells with 2C and 4C DNA content were assumed to be in G1 and G2 phase and those with intermediate DNA content in S phase of the cell cycle. The fractions of cells which had passed a given phase of the cell cycle were calculated for various times after insemination and utilized for measurements of the second and third cell cycle timing. Results of measurements for the second cell cycle: G1 phase 1.3 h, S phase 6.1 h, G2 phase 15.4 h, whereas for the third cell cycle: G1 phase 1.6 h, S phase 7.4 h, G2 phase 0.5 h. The first cleavage division was calculated as 1.6 h, the second as 1.3 h and the third as 1.2 h. Complete intra-embryonic synchronization of the DNA-synthesizing nuclei was preserved during the entire synthesis phase of 2-cell embryos, while in 4-cell embryos they were slightly asynchronized. Among mitotic cells of the first cleavage division and G1 cells of 2-cell embryos a slight interembryonic asynchronization was found which deepened during subsequent cell cycle phases.     

Induction of achromosomal cleavage by hydroxyurea in starfish embryo and the reversal by a combination of deoxyadenosine and deoxycytidine: possible involvement of salvage pathway for deoxynucleoside triphosphate biosynthesis in the presence of hydroxyurea

Effects of hydroxyurea, an inhibitor of ribonucleotide reductase, on cleavage of starfish embryos were studied. In the presence of 1 mM hydroxyurea, fertilized eggs of the starfish, Asterina pectinifera, cleaved up to the 256-cell stage and decomposed before blastulation. Before the 16-cell stage, each blastomere contained a normal nucleus or chromosomes with mitotic apparatus. The cleavage after the 16-cell stage was slow compared to the control embryos, and not all blastomeres contained a nucleus or normal chromosomes. During the fifth cell division (between 16-cell- and 32-cell-stage embryos), chromatin mass unassociated with the mitotic apparatus remained near the cleavage furrow. When hydroxyurea was removed before the 16-cell stage, the embryos developed to normal bipinnalia larvae via normal blastulae. However, the embryos were disintegrated before blastulation when hydroxyurea was removed after the 32-cell stage. DNA synthesis was normally observed before the 16-cell stage but not after the 16-cell stage, but dNTP contents in the embryos remained low throughout development in the presence of hydroxyurea. The achromosomal cleavage observed in the presence of hydroxyurea was reversed by the combination of extracellular dAR and dCR. Therefore, it is assumed that the synthesis of dNTPs required for DNA synthesis in the presence of hydroxyurea occurs via the salvage pathway using deoxynucleosides (dNR) (dNR to dNTP via dNMP and dNDP).     

Studies in vitro on mouse-egg radiosensitivity from fertilization up to the first cleavage

Super-ovulated eggs from the Balb/c strain were incubated, at various times after injection of HCG, in Whitten's medium containing tritiated thymidine. They were fixed on the following day at the 2-cell stage and prepared for autoradiography. On the basis of the results, pregnant mice were irradiated with various doses of X-rays at 15 h post HCG (fertilization), 19 h (phonuclear stage before DNA synthesis), 24 h (maximal DNA synthesis) and 27.5 h (DNA synthesis completed). On the day following irradiation, embryos were collected and classified into incleaved or 2-cell embryos, and development of the 2-cell embryos was followed in culture.

Irradiation was most effective when administered at 19 h after injection of HCG. Such a treatment increased the mortality before the first cleavage and, thereafter, from the 8-cell (100 rad) or morula stage (25, 50 rad). Blastocyst hatching and implantation were also impaired. Irradiation at other times was much less harmful for the embryos, which died mainly from the blastocyst stage. Finally, radiosensitivities of the mouse zygote at the various times studied can be estimated as follows: fertilization, + + +; pronuclear stage before DNA synthesis, + + + + +; maximal DNA synthesis, +; DNA synthesis terminated, + +.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.     

Developmental regulation of an snRNP core protein epitope during pig embryogenesis and after nuclear transfer for cloning.

The appearance and stabilization of a core protein epitope of the snRNP is developmentally regulated during pig embryogenesis. The epitope recognized by the monoclonal antibody Y12 is present in the germinal vesicle of mature oocytes and interphase nuclei of late 4-cell stage (24 to 30 hours post cleavage to the 4-cell stage) to blastocyst stage embryos. There was no antibody localization within pronuclei, or nuclei of 2-cell or early 4-cell stage embryos. Zygotes or 2-cell stage embryos cultured in the presence of alpha-amanitin to the late 4-cell stage showed no immunoreactivity, whereas control embryos had immunoreactivity. Thus antibody localization was correlated with RNA synthesis and RNA processing that begins by 24 hours post cleavage to the 4-cell stage. A final experiment showed no detectable immunoreactivity in 16-cell stage nuclei that had been transferred to enucleated activated meiotic metaphase II oocytes. Since immunoreactivity is associated with active RNA synthesis and RNA processing, it suggests that the 16-cell stage nucleus, which is RNA synthetically active, does not process RNA after nuclear transfer to an enucleated activated meiotic metaphase II oocyte.     

5-脱氧杂氮胞苷抑制小鼠附植前的胚胎发育

DNA甲基化在哺乳动物发育过程中有关键作用．在小鼠附植前胚胎发育过程中,DNA甲基化一直处于动态变化过程中．通过将体外受精胚在5-AZA-CdR中持续培养,研究5-AZA-CdR对小鼠附植前胚胎发育的影响,为附植前胚胎发育机理的研究及5-AZA-CdR的毒副作用研究提供试验基础．从原核期加入不同浓度的5-AZA-CdR时,胚胎不能发育到桑椹胚(0.2 和1.0 μmol/L)和4-细胞胚(5.0 μmol/L);从2-细胞期加入时,胚胎阻滞于未致密化的8-细胞(0.2 和1.0 μmol/L)和3/4-细胞期(5.0 μmol/L);而当从4-细胞加入时,虽然胚胎能够发育到早期桑椹胚,但发育比例同对照相比显著降低(P < 0.05)．进一步检测凋亡、基因组DNA甲基化和整体转录活性,结果显示,高浓度的5-AZA-CdR导致8-细胞和早期桑椹胚发生早期凋亡,而低浓度的5-AZA-CdR引起8-细胞和早期桑椹胚基因组DNA甲基化的降低和转录活性的降低,并且这种降低呈浓度依赖性．所以加入低浓度的5-AZA-CdR时,胚胎的DNA甲基化降低,引起转录活性的降低,进而导致胚胎发育的停滞．     

Intracytoplasmic glutathione concentration and the role of beta-mercaptoethanol in preimplantation development of bovine embryos

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of beta-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 microM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 microM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 x 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.     

Compaction rate of in vitro fertilized bovine embryos related to the interval from insemination to first cleavage

A study was conduced on early cleavage divisions and timing of compaction in bovine preimplantation-stage embryos. Zygotes were produced using conventional in vitro maturation and fertilization procedures. Twenty hours post insemination, the zygotes were denuded and cultured with oviduct epithelial cells in B2 medium + 10% estrous cow serum. Starting at 24 hours post insemination, the embryos (n=657) were evaluated every 6 hours and then were put into different co-culture drops according to their cell number. Starting from 78 hours post insemination, the cleavage rate was evaluated every 12 hours. Embryos were stained with Hoechst 33342 at the compacted morula stage or when they were degenerated, at 162 hours post insemination. Developmentally capable embryos were characterized by a rapid cleavage rate in the first 3 cell cycles and by an extended 8- to 16-cell stage. Peak concentrations of 2-, 4-, 8- and 16-cell stages emerged at 36, 42, 60 and 102 hours post insemination, respectively. Compaction did not occur until 126 hours post insemination. The rate of compaction was significantly higher in embryos that were at the 2-cell stage before or at 36 hours post insemination (P < 0.05). The mean cell numbers of compacted morulae that were identified at 126 and 138 hours post insemination were 30.9 +/- 6.8 and 31.6 +/- 7.7, respectively. These results indicate that developmentally capable bovine embryos reach the 2-cell stage at 36 hours post insemination, and that they become compacted at the 32-cell stage, which usually occurs between 126 and 138 hours post insemination.