Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the of developmental competence

Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Glucose affects monocarboxylate cotransporter (MCT) 1 expression during mouse preimplantation development

Cleavage-stage embryos have an absolute requirement for pyruvate and lactate, but as the morula compacts, it switches to glucose as the preferred energy source to fuel glycolysis. Substrates such as glucose, amino acids, and lactate are moved into and out of cells by facilitated diffusion. in the case of lactate and pyruvate, this occurs via H+-monocarboxylate cotransporter (MCT) proteins. To clarify the role of MCT in development, transport characteristics for DL-lactate were examined, as were mRNA expression and protein localisation for MCT1 and MCT3, using confocal laser scanning immunofluorescence in freshly collected and cultured embryos. Blastocysts demonstrated significantly higher affinity for DL-lactate than zygotes (K-m 20 +/- 10 vs 87 +/- 35 mmol lactate/l; P = 0.03 by linear regression) but was similar for all stages. For embryos derived in vivo and those cultured with glucose, MCT1 mRNA was present throughout preimplantation development, protein immunoreactivity appearing diffuse throughout the cytoplasm with brightest intensity in the outer cortical region of blastomeres. in expanding blastocysts, MCT1 became more prominent in the cytoplasmic cortex of blastomeres, with brightest intensity in the polar trophectoderm. Without glucose, MCT1 mRNA was not expressed, and immunoreactivity dramatically reduced in intensity as morulae died. MCT3 mRNA and immunoreactivity were not detected in early embryos. The differential expression of MCT1 in the presence or absence of glucose demonstrates that it is important in the critical regulation of pH and monocarboxylate transport during preimplantation development, and implies a role for glucose in the control of MCT1, but not MCT3, expression.

Growth hormone, insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alphaIR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alphaIR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, CH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.

Oxygen-regulated gene expression in bovine blastocysts

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

Somatic embryogenesis in sugarcane - An addendum to the invited review 'Sugarcane biotechnology: The challenges and opportunities,' in vitro cell. Dev. Biol. Plant 41(4): 345-363; 2005

Since the 1960s, numerous studies on sugarcane plant regeneration have been reported. Essentially, successful culture and regeneration of plants from protoplasts, cells, callus, and various tissue and organs, have been achieved in this crop. Although plant regeneration from callus cultures had been reported since the 1960s, definitive proof of somatic embryo development was not available until 1983. Since then, considerable progress has been made in understanding and refining somatic embryogenesis and plant regeneration in sugarcane, for which development of an efficient embryogenic system was critical for the application of transgenic technology. Recent research in Australia and South Africa has led to the development of direct somatic embryogenic systems, which may improve transgenesis in sugarcane.

The role of angiogenesis in prostate development and the pathogenesis of prostate cancer

New vessel formation, a highly-regulated, active process commencing in the embryo and evident notably during the pubertal growth spurt, is essential for normal prostate development. Reactivation of this process in response to physiological stimuli, particularly hypoxia in mature tissues, occurs with new vessels forming principally from stromal components. Although angiogenesis is complex, putatively involving a multitude of angiogenic factors and inhibitors, there is powerful evidence of the importance of the VEGF system in the development of both the normal prostate and prostate cancer. Recent advances include an understanding of how castration acts through the VEGF system to inhibit angiogenesis. Stromal-endothelial and epithelial-endothelial interactions are just beginning to be investigated. A better understanding of how physiological angiogenesis is controlled should help to provide further insights into the mechanism of disregulated angiogenesis in tumours. Ultimately, new antiangiogenic agents are likely to find a role in the management of patients with prostate cancer.

The role of insulin-like growth factor II and its receptor in mouse preimplantation development

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.

Desperately seeking donors: The 'saviour siblings' decision in quintaville v human fertilisation embryology authority (UK)

The recent House of Lords decision in Quintavalle v Human Fertilisation and Embryology Authority has raised difficult and complex issues regarding the extent to which embryo selection and reproductive technology can be used as a means of rectifying genetic disorders and treating critically ill children. This comment outlines the facts of Quintavalle and explores how the House of Lords approached the legal, ethical and policy issues that arose out of the Human Fertilisation and Embryology Authority's (UK) decision to allow reproductive and embryo technology to be used to produce a 'saviour sibling' whose tissue could be used to save the life of a critically ill child. Particular attention will be given to the implications of the decision in Quintavalle for Australian family and medical law and policy. As part of this focus, the comment explores the current Australian legislative and policy framework regarding the use of genetic and reproductive technology as a mechanism through which to assist critically ill siblings. It is argued that the present Australian framework would appear to impose significant limits on the medical uses of genetic technology and, in this context, would seem to reflect many of the principles that were articulated by the House of Lords in Quintavalle.

Apoptotic cell removal in development and tissue homeostasis

Cell deletion is a physiological process for the development and maintenance of tissue homeostasis in metazoa. This is mainly achieved by the induction of various forms of programmed cell death followed by the recognition and removal of the targeted cells by phagocytes. In this review, we will discuss cell deletion in relation to the development and function of the innate immune system, particularly of the mononuclear phagocyte system (MPS), its ontogeny and potential role in tissue remodeling in the embryo and adult. Ongoing studies are addressing the roles of professional phagocytes of the MPS and neighboring tissue cells in dying cell removal, and candidate molecules that might attract mononuclear phagocytes to the dying cells. The potential phagocyte must discriminate between living and dying cells; current concepts for this discrimination derive from the observation of newly exposed ligands on the dying cells and new evidence for direct inhibition of uptake by viable cells.

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass. notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tall and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tall. Imo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Scube2 mediates Hedgehog signalling in the zebrafish embryo

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the you-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian, gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction. (c) 2006 Elsevier Inc. All rights reserved.

The makings of maleness: towards an integrated view of male sexual development

As the mammalian embryo develops, it must engage one of the two distinct programmes of gene activity, morphogenesis and organogenesis that characterize males and females. In males, sexual development hinges on testis determination and differentiation, but also involves many coordinated transcriptional, signalling and endocrine networks that underpin the masculinization of other organs and tissues, including the brain. Here we bring together current knowledge about these networks, identify gaps in the overall picture, and highlight the known defects that lead to disorders of male sexual development.

Development of a cell culture method for quantal assay of strain I-2 of Newcastle disease virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain 1-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain 1-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain 1-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.