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PubMed2003

Exposure of bovine oocytes to the endogenous metabolite 2-methoxyestradiol during in vitro maturation inhibits early embryonic development.

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

2-Methoxyestradiol↗
PubMed1999

mRNA and protein localization of the IGF system during mouse embryonic development in areas with apoptosis.

We analysed mRNA and protein localization of the IGF system components in regions with apoptosis during mouse development between 9.5 and 13.5 days post coitum. A spatio-temporal relationship between these expression patterns and the onset of apoptosis in specific areas was sought. The IGFBP mRNA and protein expression patterns were tissue-specific. In most tissues, mRNA expression patterns colocalized with protein localization. Discrepancies between mRNA and protein detection were found in, for example, lens, neural layer of the retina, whiskers and somites. Localization of the IGFs, the type I IGF receptor and IGFBP-2 correlated well with cell death regions. When these genes were expressed no apoptosis occurred and vice versa. Correlation of IGFBP-3, -4 and -5 with apoptosis regions was noticed only at 13.5 days post coitum. In eye muscles, whiskers and somites, the expression of IGF system components preceded the occurrence of apoptotic cells. When IGF-I expression ceased, apoptosis occurred in these areas. In conclusion, our results suggest that IGF-I, the type I IGF receptor and IGFBP-2 inhibit apoptosis. In contrast, IGFBP-3, -4 and -5 may stimulate apoptosis by trapping the IGFs. Tissue-specific modulation of IGF protective action against apoptosis by the different IGFBPs during mouse embryonal development may contribute to organ specific morphology.

Animals↗
PubMed2000

Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development.

pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165-171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021-1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301-2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.

Animals↗
PubMed1993

The role of a Drosophila POU homeo domain gene in the specification of neural precursor cell identity in the developing embryonic central nervous system.

The Drosophila embryonic central nervous system (CNS) is derived from a stereotypic array of progenitor stem cells called neuroblasts (NBs). Each of the approximately 25 NBs per hemisegment undergoes repeated asymmetric divisions to produce, on average, 5-10 smaller ganglion mother cells (GMCs); each GMC, in turn, divides to produce two neurons. We demonstrate that the protein product encoded by a POU homeo domain gene (dPOU28/pdm-2) is expressed in the cell nuclei of a subset of NBs and GMCs. In the wild-type animal, GMC-1 is the only identified cell in the NB4-2 lineage that expresses dPOU28 protein to a high level, and it divides to produce the RP2 neuron and a second cell of unknown fate. Our results suggest that the presence of ectopically induced dPOU28/pdm-2 protein in the progeny of GMC-1 is sufficient to cause both of these cells to adopt their parental GMC-1 cell fate, leading to duplication of the RP2 neuron (and its sister cell) on the basis of both immunological and morphological criteria. These observations clearly implicate a role for dPOU28/pdm-2 in the specification of GMC-1 cell identity in the NB4-2 lineage and possibly in the specification of cell fate in other NB lineages in the developing embryonic CNS.

Animals↗
PubMed2001

Utilisation of lipids, protein, ions and energy during embryonic development of Australian oviparous skinks in the genus Lampropholis.

The contents of eggs and neonates of the Australian skinks, Lampropholis guichenoti and L. delicata, are described and compared to allow interpretation of nutrient utilisation by the developing embryo. Even though the females are the same size, L. guichenoti lay smaller clutches of larger eggs (egg contents=41.6+/-1.2 mg dry mass) than L. delicata (26.6+/-2.8 mg). The energy density is the same for eggs (30.5+/-0.9 J/g ash-free dry mass for L. guichenoti and 29.9+/-1.1 J/mg for L. delicata) and neonates (22.5+/-1.3 J/mg for L. guichenoti and 23.5+/-0.4 J/mg for L. delicata) between species. The amount of nitrogen (protein) in neonates is only slightly lower than that in eggs, whereas there is a large and significant decline in total lipids. Thus, like some other skinks, protein is a source of metabolic energy during embryogenesis, although not as important as lipid. Triacylglycerol is the major lipid component of the eggs (80% of total lipid), with phospholipid forming only approximately 10% of the total lipid. The fatty acid profile of the phospholipid is distinguished by a high proportion of arachidonic acid (8%), a significant proportion of eicosapentaenoic acid (2-4%) and a relatively low proportion of docosahexaenoic acid (2-3%) compared to chickens. Eggs of both species have remarkably low concentrations of free cholesterol compared to other amniote eggs (0.7% for L. guichenoti and 1.3% for L. delicata). The loss of lipid during embryonic development is almost entirely due to the selective utilisation of yolk triacylglycerol, presumably for energy. By contrast, the amount of phospholipid recovered from the neonates was the same as that originally in the eggs. Moreover, significantly more total cholesterol was present in the neonates than in the eggs, suggesting that biosynthesis of additional cholesterol occurred during development. The phospholipids of the neonates contain higher proportions of arachidonic (11-12%) and docosahexaenoic (8%) acids than the phospholipids of the eggs. Eicosapentaenoic acid is less prevalent in phospholipids in neonates than in eggs. Neonates of both species contain significantly more calcium than the fresh egg contents (L. guichenoti, eggs 0.303+/-0.051 mg, neonates 0.641+/-0.047 mg; L. delicata, eggs 0.187+/-0.013 mg, neonates 0.435+/-0.033 mg), presumably as a result of resorption of calcium from the eggshell. Interestingly, there is also significantly more sodium in neonates than in the contents of fresh eggs (L. guichenoti, eggs 0.094+/-0.010 mg, neonates 0.184+/-0.011 mg; L. delicata, eggs 0.084+/-0.011 mg, neonates 0.151+/-0.010 mg). There is no significant difference in the content of potassium and magnesium in eggs and neonates of either species. Although the fresh eggs of L. delicata have a significantly higher sodium concentration than L. guichenoti, there is no difference in the concentrations of calcium, magnesium, potassium or sodium in the neonates of the two species.

Animals↗
PubMed2004

Cloning of a novel phospholipase C-delta isoform from pacific purple sea urchin (Strongylocentrotus purpuratus) gametes and its expression during early embryonic development.

Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, including fertilization and development of the embryo. One of the key mechanisms involved in triggering intracellular calcium release is the generation of the second messenger inositol-1,4,5-phosphate (IP(3)) by the phospholipase C (PLC) class of enzymes. Although five distinct forms of PLC have been identified in mammals (beta, gamma, delta, epsilon, and zeta), only one, PLCgamma, has thus far been detected in echinoderms. In the present study, we describe the isolation of a cDNA encoding a novel PLC isoform of the delta (delta) subclass, PLC-deltasu, from the egg of the Pacific purple sea urchin Strongylocentrotus purpuratus. We also demonstrate the presence of this PLC within the sperm and in the early embryo. The PLC-deltasu cDNA (2.44kb) encodes a 742 amino acid polypeptide with an open reading frame of 84.6kDa and a pI of 6.04. All of the characteristic domains found in mammalian PLCdelta isoforms (PH domain, EF hands, an X-Y catalytic region, and a C2 domain) are present in PLC-deltasu. A homology search revealed that PLC-deltasu shares most sequence identity with bovine PLCdelta2 (39%). We present evidence that PLC-deltasu is expressed in unfertilized eggs, fertilized eggs, and in the early embryo. In addition to Northern and polymerase chain reaction (PCR) analyses, in situ hybridization experiments further demonstrated that the embryonic regions within which the PLC-deltasu transcript can be detected during early embryonic development are associated with the highest levels of proliferative activity, suggesting a possible involvement with metabolism or cell cycle regulation.

Amino Acid Sequence↗
PubMed1996

In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development.

Regulatory effect of GH on follicular growth and development in the cow is well documented. The aim of this study was to investigate the role of GH on in vitro bovine oocyte maturation. Therefore bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of 10, 100, or 1,000 ng/ml bovine GH (NIH-GH-B18). The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air and nuclear stage was assessed after 2, 4, 8, 16, 22, and 24 hr of incubation using DAPI staining. To assess the effect of GH on developmental capacity of the oocytes, COCs were incubated in the presence of GH for 22 hr, followed by IVF and in vitro embryo culture. Cultures without GH served as controls. For subsequent development, the embryos were cultured in M199 supplemented with 10% FCS on a monolayer of BRL cells. Embryos were scored morphologically and the efficiency of the culture system was evaluated as (1) the percentage of cleaved embryos 4 days after IVF, (2) the percentage of blastocysts on day 9 expressed on the basis of the number of oocytes at the onset of culture, and (3) the percentage of hatched blastocysts on day 11 expressed on the basis of the total number of blastocysts present at day 9. GH (100 and 1,000 ng/ml) significantly accelerated nuclear maturation (P < 0.001). At 4 and 8 h the percentage of oocytes in GV stage after GH treatment (54% and 19%) was significantly lower than the control (64% and 41%). Similarly at 16 and 22 h the percentage of oocytes in MII stage was significantly higher in the GH-treated group; (58% and 77%) and (46% and 62%) for GH and control respectively. The number of oocytes in MII beyond 22 hr of culture did not differ; 100 and 1,000 ng/ml GH induced significant cumulus expansion (P < 0.05), which was not observed in the absence of GH. Addition of 100 and 1,000 ng/ml GH during maturation significantly (P < 0.01) enhanced subsequent cleavage rate from (64% and 67%) in control to (75% and 81%) in GH-treated group; embryonic development in terms of day 9 blastocyst formation was also significantly increased in the presence of GH (29% and 34%) compared to the control (18% and 24%). The hatchability of the blastocysts was not influenced by GH. From the present data, it can be concluded that GH present during IVM has a beneficial effect on subsequent development.

Animals↗
PubMed2005

Fertilization regulates apoptosis of Ciona intestinalis extra-embryonic cells through thyroxine (T4)-dependent NF-kappaB pathway activation during early embryonic development.

In Ciona intestinalis, the elimination of extra-embryonic test cells during early stage of development is delayed by a fertilization signal. Test cells undergo a caspase-dependent apoptosis event repressed by thyroxine (T4)-activated NF-kappaB. When apoptosis was experimentally blocked, the hatching stage was delayed. The incubation of unfertilized eggs with a 1-h-fertilized egg extract or purified T4 restored apoptosis in test cells at a similar timing than found in fertilized eggs. Ciona expresses specific genes forming a functional IkappaB/NF-kappaB pathway. One, Ci-p65, was transiently induced upon fertilization via T4 and found to exert its anti-apoptotic role in test cells nuclei as well as in a reconstituted cell system. Blocking NF-kappaB activity by dexamethasone-induced overexpression of Ci-IkappaB abrogated the repression of apoptosis in test cells. Overall, the data are consistent for defining a central coupling role of both T4 and NF-kappaB during early embryo development.

Amino Acid Sequence↗
PubMed2000

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

Alleles↗
PubMed1996

Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development.

Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.

Amino Acid Sequence↗