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PubMed2006

Profilin and actin-related proteins regulate microfilament dynamics during early mammalian embryogenesis.

BACKGROUND: Profilins are ubiquitous proteins widely distributed in animals, including humans. They regulate actin polymerization by sequestering actin monomers in association with other actin-related proteins (Arps). Actin remodelling is essential for oocyte maturation, fertilization and embryo development; yet the role of profilins in these events is not well understood. Here we investigate profilin distribution and function during bovine fertilization and early embryogenesis, and we examine profilin localization with respect to the co-distribution of other Arps. METHODS AND RESULTS: Western blotting, confocal microscopy with immunofluorescence and protein inhibition studies with antibodies were implemented. Profilin distributes inside interphase nuclei, throughout the cytoplasm and near the cell cortex at different stages of bovine oocyte maturation, fertilization and embryo development. Expression is detected through the blastocyst stage, where profilin localizes to the inner cell mass as well as trophectoderm. Profilin co-distributes with actin monomers and Arps vasodilator-stimulated phospho protein, p140mDia, Arp 3 and p80 coilin in pronucleate-stage zygotes. Antiprofilin antibodies inhibit normal embryo development by disrupting microfilaments, but not microtubules, and result in a higher concentration of profilin and p140mDia mislocalized to the cortex. CONCLUSIONS: These findings demonstrate that profilin regulates actin dynamics both within the cytoplasm and inside the nuclei of developing mammalian embryos and that its function is essential during fertilization to ensure successful development.

Actin Cytoskeleton↗
PubMedDate not supplied

Mitochondria: potential roles in embryogenesis and nucleocytoplasmic transfer.

This review examines current understanding of mammalian mitochondria and mitochondrial DNA in the light of new reproductive technologies. Mitochondria are central to ageing, apoptosis, metabolism and many diseases. They are controlled by a dual genome system, with cooperation between endogenous mitochondrial genes and mitochondrial genes translocated to the nucleus over the course of evolution. This translocation has been accompanied by extreme compression of the mitochondrial genome, with little tolerance for mutations or heteroplasmy (multiple genomes). The highly compact mitochondrial genome appears to be maintained by a stringent numerical bottleneck in embryogenesis and oogenesis, followed by clonal expansion from a highly selected subset of precursor molecules. The dual nature of control between nucleus and cytoplasm sets up potential conflicts, which are normally resolved by natural selection. Such potentially opposing interests and mechanisms are probably partly to blame for the poor rates of success in cloning animals by nuclear transfer. The ability to construct cell systems and animal embryos with novel combinations and permutations of nuclear and cytoplasmic genes will provide powerful tools for examining these fundamental biological questions. Clinically, attempts to 'rescue' abnormal human oocytes or embryos by cytoplasmic transfer risk complex and unpredictable outcomes emerging from disharmonious nuclear-cytoplasmic interactions.

Aging↗
PubMed2000

Polarity and signalling in plant embryogenesis.

The establishment of the apical-basal axis is a critical event in plant embryogenesis, evident from the earliest stages onwards. Polarity is evident in the embryo sac, egg cell, zygote, and embryo-suspensor complex. In the embryo-proper, two functionally distinct meristems form at each pole, through the localized expression of key genes. A number of mutants, notably of the model genetic organism Arabidopsis thaliana, have revealed new gene functions that are required for patterning of the apical-basal axis. There is now increasing evidence that two particular modes of signalling, via auxin and cell wall components, play important roles in co-ordinating the gene expression programmes that define determinative roles in the establishment of polarity.

Arabidopsis↗
PubMed2005

In situ expression of two storage protein genes in relation to histo-differentiation at mid-embryogenesis in Medicago truncatula and Pisum sativum seeds.

The seed consists of several layers of specialized cell-types that divide and differentiate following a highly regulated programme in time and space. A cytological approach was undertaken in order to study the histo-differentiation at mid-embryogenesis in Medicago truncatula as a model legume, and in Pisum sativum using serial sections of embedded immature seed. Little published information is available about seed development in Medicago species. The observations from this study revealed a number of distinctive features of Medicago seed development and differentiation. Transfer cells, involved in nutrient transfer to the embryo, were clearly identified in the thin-walled parenchyma of the innermost integument. Histological Schiff-naphthol enabled carbohydrate accumulation to be followed in the different seed compartments, and revealed the storage protein bodies. Non-radioactive mRNA in situ hybridization, was carried out using mRNA probes from two highly expressed genes encoding the major vicilin and legumin A storage protein types. The timing of mRNA expression was related to that of the corresponding proteins already identified.

Cell Differentiation↗
PubMed2002

Epigenetic marks at BRCA1 and p53 coding sequences in early human embryogenesis.

In the vertebrate genome, methylation of deoxycytosine residues of CpGs dinucleotide has been associated with transcriptional silencing of genes, parental imprinting, X-inactivation and chromatin remodelling. In human somatic tissues, the 5' end of the BRCA1 CpG island is methylated, whereas this region is unmethylated in mature germ cells and early embryos. In gametes, as in somatic tissues, the CpG sites in the coding region are methylated. We took advantage of this bimodal distribution as a model to analyse the epigenetic reprogramming of coding regions during early human embryogenesis using the bisulphite-based genomic sequencing method. During preimplantation divisions, exon 11 of BRCA1 was slowly demethylated and retained approximately 30% of its methylated residues at the blastocyst stage. Moreover, the change in the distribution of methylated residues was not restricted to the BRCA1 gene, since for another gene, p53, a relatively high level of methylation (50%) of exon 4 was observed in blastocysts. Taken together, these data suggest that a significant part of the methylated residues of coding sequences might be conserved during preimplantation development.

Animals↗
PubMed1991

Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis.

We have isolated a cDNA clone encoding the Xenopus homologue of the transcription factor AP-2 (XAP-2). The predicted amino acid sequence derived from the Xenopus cDNA shows very strong conservation with the amino acid sequence of human AP-2, suggesting that this protein is evolutionarily conserved, at least among vertebrates. This is further substantiated by the demonstration that an in vitro translation product of XAP-2 cDNA bound specifically to an AP-2 binding site from the human MT-IIA gene. Northern blot analysis of Xenopus embryo RNA revealed the existence of three major XAP-2 mRNA species that were only detectable after the midblastula transition (when embryonic transcription is activated), with peak accumulation of the transcripts occurring during gastrulation. Therefore, in contrast to other Xenopus transcription factors, XAP-2 is not maternally derived but arises exclusively from zygotic transcription. Unlike the situation in cultured human teratocarcinoma (NT2) cells, retinoic acid treatment did not induce XAP-2 mRNA in Xenopus embryos, even though the treatment had a pronounced morphogenetic effect on the embryos. Our results suggest that XAP-2 may play a distinctive role during Xenopus embryogenesis.

Amino Acid Sequence↗
PubMed1995

Xenopus sonic hedgehog as a potential morphogen during embryogenesis and thyroid hormone-dependent metamorphosis.

The hedgehog family of proteins have been implicated as important signaling molecules in establishing cell positional information and tissue patterning. Here we present the cloning and characterization of a hedgehog homologue from Xenopus laevis similar to the sonic class of vertebrate hedgehog genes. We isolated Xenopus hedgehog (Xhh) from a subtractive hybridization screen designed to identify genes induced by thyroid hormone during metamorphosis of the X.laevis gastrointestinal tract. In the intestine, Xhh mRNA expression was up-regulated at the climax of metamorphosis (stage 62) when intestinal epithelium underwent morphogenesis. Treatment of pre-metamorphic tadpoles with exogenous thyroid hormone (TH) resulted in a similar pattern of Xhh induction. Furthermore, TH induction was resistant to inhibitors of protein synthesis suggesting that Xhh is a direct thyroid hormone response gene. The expression and TH regulation of Xhh was not limited to the intestine, but was also observed in the limb and a mixture of pancreas and stomach. Throughout development, Xhh mRNA was present at varying levels with the earliest expression being detected at neurula stage. The highest levels of Xhh were observed between stages 33 and 40 shortly before tadpole feeding begins. Whole mount in situ hybridization analysis of Xhh expression in pre-hatching, stage 32 tadpoles demonstrated staining in the notochord and floor plate similar to that observed for other vertebrate hedgehog genes. Together, these data suggest a putative role for Xhh in organ development during both amphibian embryogenesis and metamorphosis.

Aging↗
PubMed2000

Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis.

The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is 'masked' shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca(2+)-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.

Amino Acid Sequence↗
PubMed1989

Growth factors in embryogenesis.

The growing appreciation that embryonic material is a rich source of growth factors underlies increasing interest in the role of growth factors in early development. These developments have arisen from a desire to understand fundamental embryological processes; the control of differentiation and the generation of form. One expectation of this research is the discovery of new forms of regulatory agent with novel biological and therapeutic potential. Furthermore, there is a direct relationship between growth and differentiation processes in early development and pathological processes in the adult; for example, the behaviour of tumour cells has historically been compared to that of embryonic cells and regenerative processes in the adult such as angiogenesis, wound healing and fracture repair may be considered to recapitulate events that occur in the normal course of embryogenesis. An understanding of the mechanisms that underlie these fundamental processes in normal development may therefore lead to a concomitant understanding of related pathological events in the adult.

Animals↗
PubMed1994

Timing of development is a critical parameter for predicting successful embryogenesis.

Development of embryos from the 1-cell stage into blastocysts in vitro is generally slower than the time-course for development in vivo. It was the objective of this work to determine whether embryos that reach the 8-cell stage within a normal time-frame have a developmental advantage (both in vitro and post-embryo transfer) over slower embryos. Hamster 1-cell embryos were collected 10 h post-egg activation (PEA) and cultured for 48 h (58 h PEA = t50 for 8-cell embryo development in vivo) in hamster embryo culture medium-6. Embryos were sorted according to stage reached, culture was continued in fresh medium and stage of development was observed at 78, 82 and 86 h PEA. At 58 h PEA, embryos were < 4-cell (4%), 4-cell (19%), 5- to 7-cell (16%) or 8-cell (61%). The 58 h 8-cell embryos had a significantly greater ability to develop to the blastocyst stage than 58 h 4-cell embryos at 78, 82 and 86 h PEA (74 versus 13%, 69 versus 25% and 65 versus 37% respectively). The percentages of 14-day-old fetuses collected after embryo transfer indicated that morulae and blastocysts derived from 58 h 4-cell embryos were, on average, less viable (26% fetuses) than morulae and blastocysts from 58 h 8-cell embryos (51% fetuses). Thus morulae and blastocysts developing in vitro from faster or slower cleaving embryos can be qualitatively as well as quantitatively different. These data indicate that the timing of development in vitro, specifically the timing of completion of the third cell cycle, is a critically important parameter for predicting successful embryogenesis in the hamster.

Animals↗
PubMed1999

Molecular cloning and characterization of two zebrafish alpha(1,3)fucosyltransferase genes developmentally regulated in embryogenesis.

Some alpha(1,3)fucosylated oligosaccharides serve as counter receptors to lectin-like adhesion proteins or are expressed with temporal precision during embryogenesis, and alpha(1, 3)fucosyltransferase is a key enzyme in the production of these oligosaccharides. Two alpha(1,3)-fucosyltransferase genes, designated zFT1 and zFT2, were cloned from zebrafish. Sequence comparisons with other genes indicated that zFT1 and zFT2 share about 30% amino acid sequence identity with human alpha(1, 3)fucosyltransferases. Although the alpha(1,3)fucosyltransferases cloned so far can be classified into three types-myeloid, Lewis, and leukocyte-by virtue of their amino acid sequences, phylogenetic analysis indicated that neither zFT1 nor zFT2 belongs to any of these categories. The expression of zFT1 or zFT2 in mammalian cells induces alpha(1,3)fucosyltransferase activity to synthesize the Lewis x structure from pyridylaminated lacto-N-neotetraose; however, lacto-N-tetraose does not serve as a substrate. Reverse transcriptase-polymerase chain reaction analysis revealed that zFT1 is transcribed during a restricted period before hatching, whereas the mRNA for zFT2 was detected only after hatching.

Amino Acid Sequence↗
PubMed1999

Inactivation of proprotein convertase, PACE4, by alpha1-antitrypsin Portland (alpha1-PDX), a blocker of proteolytic activation of bone morphogenetic protein during embryogenesis: evidence that PACE4 is able to form an SDS-stable acyl intermediate with alpha1-PDX.

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.

Animals↗
PubMed1995

Midkine (MK) expression in extraembryonic tissues, amniotic fluid, and cerebrospinal fluid during mouse embryogenesis.

Midkine (MK) and pleiotrophin (PTN) constitute a new family of heparin-binding growth factors. Extraembryonic membranes and the placenta of the mouse expressed MK mRNA at 11.5 days gestation. While the MK mRNA level in extraembryonic membranes decreased during embryogenesis, that in the placenta remained unchanged. Immunohistochemical studies showed that Mk was located in the yolk sac and in the amnion at 11.5 days gestation. PTN mRNA expression was weak in extraembryonic membranes and was scarcely detectable in the placenta. Western blot analysis revealed the presence of MK in amniotic fluid and cerebrospinal fluid, in amounts of more than 1 microgram/ml, raising the possibility that MK delivered by these fluids participates in the regulation of organogenesis. Transport of MK from the site of its synthesis appears to also occur in the adult kidney, since MK mRNA and the MK protein are localized in different regions of the kidney.

Amino Acid Sequence↗
PubMed2002

OSTF1: a HD-GL2 family homeobox gene is developmentally regulated during early embryogenesis in rice.

In many eukaryotic organisms, homeobox genes are important regulators that specify the cell fate and body plan in early embryogenesis. In this study, a gene designated OSTF1 (Oryza sativa transcription factor 1) encoding a homeodomain protein in rice was isolated and characterized. The encoded OSTF1, although sharing only approximately 51% sequence identity with other HD-GL2 members, contains four characteristic motifs (an N-terminal acidic region, a homeodomain, a truncated leucine zipper, and a START domain). OSTF1 was detected as a single copy gene in rice. The transcripts were absent in young panicle or mature spikelet before anthesis, but appeared very early in the pollinated grain with a transient profile. In vegetative tissues examined, expression was only detectable in root. In situ hybridization analysis on developing grains revealed that OSTF1 was strongly and uniformly expressed in the embryo at the globular stage and preferentially localized to the protoderm at 3-6 d after pollination. Expression was also detectable in the integument and throughout the endosperm. Although OSTF1 is not closely related to the remaining HD-GL2 members in sequences, this gene exhibits an analogous epidermis-preferential expression pattern.

Amino Acid Sequence↗
PubMed2000

Influencing micropropagation and somatic embryogenesis in mature trees by manipulation of phase change, stress and culture environment.

This review focuses on clonal propagation of mature trees by tissue culture. Most trees have marked phase changes that result in a decline in their potential for somatic embryogenesis or micropropagation. By altering conditions of the source material ex vitro, or by changing in vitro conditions encountered by the explant, rejuvenation and increased propagation can sometimes be accomplished. Various methods of enhancing micropropagation are reviewed, with particular emphasis on manipulations that involve application of osmotic, temperature or hormonal stress.

Culture Media↗
PubMed1991

Storage protein changes during zygotic embryogenesis in interior spruce.

The major storage proteins isolated from protein bodies of embryo tissues of interior spruce Picea glauca (Moench) Voss/Picea engelmanii Parry had apparent molecular weights of 41, 35, 33, 24 and 22 kD. Minor proteins of 30 and 27.5 kD were also observed. Based on their solubility characteristics, the 41 kD protein was identified as a water and buffer-soluble albumin, and the 35, 33, 24 and 22 kD proteins were characterized as buffer-insoluble, high salt-soluble globulins. Two-dimensional electrophoresis revealed each protein was composed of several isoelectric variants. Developmentally specific accumulation of storage proteins was observed during embryogenesis. The 41 kD protein only accumulated during the later stages of cotyledon maturation, whereas the other storage proteins began to accumulate during the early stages of embryo development. All storage proteins showed major accumulations during cotyledon maturation.

Journal Article↗
PubMed2002

CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage.

Regulated movement of cellular factors between the cytoplasm and nucleus is required for fundamental cellular processes ranging from cell cycle control to transcriptional regulation. CRM1 is a nuclear export factor whose function is to actively transport nuclear cargos that bear nuclear export sequences to the cytoplasm. Because CRM1 likely plays a role in the intracellular regulation of many cellular processes, we set out to characterize CRM1 function during early mammalian embryogenesis. A series of embryo culture experiments that employed a specific inhibitor of CRM1, leptomycin B, indicated that CRM1 function is not required for development until after the 4-cell stage of porcine embryo development. Immunolocalization of CRM1 in fixed embryos revealed that CRM1 is localized in a unique pattern during the period of time when the embryo does not have a developmental requirement for CRM1. Despite these findings, a microinjection assay showed that CRM1 function persists during this period of development. This demonstrates that although CRM1 is present in a functional form throughout mammalian embryo development, its function is not required for early cleavage.

Animals↗
PubMed1983

Effects of chronic administration of delta-9-tetrahydrocannabinol on early embryogenesis of the mouse.

The effects of chronic administration of delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marihuana, on embryogenesis were investigated in the mouse treated with THC prior to sexual maturation. The study was designed to imitate the prolonged abuse of marihuana by sexually immature and adolescent girls. Female Swiss-Webster mice were injected, intraperitoneally, with THC 5 mg/kg per day or with the vehicle for 21 consecutive days, starting on the 30th day of life, which is prior to their sexual maturation. The dose of THC was approximately equivalent to that absorbed by a man smoking 1 to 2 marihuana cigarettes per day. Superovulation was then induced, and the mice were mated with untreated males. Ova were recovered from the oviducts just prior to the first cleavage division, and at about the time of the second cleavage division. The incidence of degenerative ova increased from 13.6% in the controls to 19.1% in the THC-treated group (P less than 0.001) at the time of the second cleavage division, but there was no difference in the incidence of morphologically abnormal ova between the two groups just prior to the first cleavage division. The rate of successful completion of the first cleavage division, but not the second cleavage division, was adversely affected by THC, resulting in accumulation of morphologically abnormal ova at the later stage. In view of the known disruptive effect of THC on the process of cell division, it was postulated that THC might have adversely affected meiosis, leading to the inability of the ova to undergo the first cleavage division.

Animals↗