X-linked α-thalassemia with mental retardation is downstream of protein kinase A in the meiotic cell cycle signaling cascade in Xenopus oocytes and is dynamically regulated in response to DNA damage†.

X-linked α-thalassemia with mental retardation (ATRX) is a chromatin remodeling protein that belongs to the SWItch/sucrose non-fermentable (SWI2/SNF2) family of helicase/ATPases. During meiosis, ATRX is necessary for heterochromatin formation and maintenance of chromosome stability in order to ensure proper assembly of the metaphase II spindle. Previously, we established ATRX as a novel progesterone regulated protein during bovine meiotic maturation, in addition to being dynamically regulated in response to DNA damage in oocytes. In the present study, we utilize the Xenopus laevis model system to further elucidate the signaling pathways regulating ATRX expression within the oocyte. Here, we present an analysis of endogenous ATRX protein expression during oogenesis, oocyte meiotic maturation, and early embryonic development. ATRX expression is dynamically regulated as evidenced by loss of the protein in metaphase II of meiosis. The downstream activation of meiosis via protein kinase A inhibition resulted in a similar decrease in ATRX protein expression. We demonstrate that the ATRX protein is detected in ubiquitin immuno-precipitates from germinal vesicle oocyte extracts and experimentally demonstrate that proteosomal degradation is responsible for the decreased expression of ATRX during meiosis. ATRX expression is significantly increased in response to gamma-irradiation induced DNA damage in oocytes and embryos. This increased expression is independent of p53 protein expression in apoptotic embryos, as determined by the expression of active caspase-3. Thus, regulation of ATRX protein expression impacts on G2-M progression and ultimately has consequences for cell survival.

ATRX is a novel progesterone-regulated protein and biomarker of low developmental potential in mammalian oocytes.

A multi-species meta-analysis of published transcriptomic data from models of oocyte competence identified the chromatin remodelling factor ATRX as a putative biomarker of oocyte competence. The objective of the current study was to test the hypothesis that ATRX protein expression by cumulus-oocyte complexes (COCs) reflects their intrinsic quality and developmental potential. In excess of 10,000 bovine COCs were utilised to test our hypothesis. COCs were in vitro matured (IVM) under conditions associated with reduced developmental potential: IVM in the presence or absence of (1) progesterone synthesis inhibitor (Trilostane); (2) nuclear progesterone receptor inhibitor (Aglepristone) or (3) an inducer of DNA damage (Staurosporine). ATRX protein expression and localisation were determined using immunocytochemistry and Western blot analysis. A proportion of COCs matured in the presence or absence of Trilostane was in vitro fertilised and cultured, and subsequent embryo development characteristics were analysed. In addition, ATRX expression was investigated in 40 human germinal vesicle-stage COCs. Our results showed that ATRX is expressed in human and bovine germinal vesicle oocytes and cumulus cells. In bovine, expression decreases after IVM. However, this decline is not observed in COCs matured under sub-optimal conditions. Blastocyst development rate and cell number are decreased, whereas the incidence of abnormal metaphase phase spindle and chromosome alignment are increased, after IVM in the presence of Trilostane (P < 0.05). In conclusion, localisation of ATRX to the cumulus cell nuclei and oocyte chromatin, after IVM, is associated with poor oocyte quality and low developmental potential. Furthermore, ATRX is dynamically regulated in response to progesterone signalling.

Progesterone regulation of AVEN protects bovine oocytes from apoptosis during meiotic maturation.

Inhibition of progesterone (P4) synthesis by cumulus cells during bovine in vitro oocyte maturation (IVM) causes a decrease in subsequent embryo development, indicating that P4 intracellular signaling within the cumulus oocyte complex (COC) is important for oocyte developmental competence. The aim of the present study was to further elucidate, on a protein level, the downstream signaling pathway involved in P4 regulation of oocyte developmental competence. COCs were subjected to IVM for 24 h in the presence or absence of trilostane, aglepristone, or promegestone (R5020). These altered IVM conditions resulted in dynamic changes in protein expression of the progesterone receptors and the cell death-regulated proteins AVEN, BCL-xL, and active caspase 3. In addition, AVEN protein localization, caspase 3 activation, and mitochondrial distribution were studied by immunofluorescence. Inhibition of progesterone synthesis (trilostane treatment) resulted in changes in AVEN localization within the COC, corresponding to caspase 3 activation and altered mitochondrial distribution. AVEN was also found to bind BCL-xL in COCs, but this interaction was lost following treatment with trilostane.

Developmental competence in oocytes and cumulus cells: candidate genes and networks.

Common aspects of infertility can be seen across several species. In humans, dairy cows, and mares there is only a 25-35% chance of producing a live offspring after a single insemination, whether natural or artificial. Oocyte quality and subsequent embryo development can be affected by factors such as nutrition, hormonal regulation, and environmental influence. The objective of this study was to identify genes expressed in oocytes and/or cumulus cells, across a diverse range of species, which may be linked to the ability an oocyte has to develop following fertilization. Performing a meta-analysis on previously published microarray data on various models of oocyte and embryo quality allowed for the identification of 56 candidate genes associated with oocyte quality across several species, 4 of which were identified in the cumulus cells that surround the oocyte. Twenty-one potential biomarkers were associated with increased competence and 35 potential biomarkers were associated with decreased competence. The upregulation of Metap2, and the decrease of multiple genes linked to mRNA and protein synthesis in models of competence, highlights the importance of de novo protein synthesis and its regulation for successful oocyte maturation and subsequent development. The negative regulation of Wnt signaling has emerged in human, monkey, bovine, and mouse models of oocyte competence. Atrx expression was linked to decreased competence in both oocytes and cumulus cells. Biological networks and transcription factor regulation associated with increased and decreased competence were also identified. These genes could potentially act as biomarkers of oocyte quality or as pharmacological targets for manipulation in order to improve oocyte developmental potential.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants.

BACKGROUND: Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. RESULTS: Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. CONCLUSIONS: We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

Cloning and characterization of Xenopus laevis Smac/DIABLO.

Mitochondria-mediated apoptosis plays a central role in animal development and tissue homeostasis, and mitochondria contain several pro-apoptotic proteins that have key roles in apoptosis. Smac/DIABLO was identified as a mitochondrial protein that is released into the cytosol following apoptotic stimuli, subsequently blocking the anti-apoptotic activity of inhibitor of apoptosis proteins. Through expressed sequence tag (EST) analysis we detected evidence for the presence of a number of Xenopus counterparts to mammalian mitochondrial pro-apoptotic proteins. EST and genome sequencing provides evidence for the presence of endonuclease G, AIF, HtrA/Omi and Smac/DIABLO in Xenopus laevis and tropicalis. Here we report the cloning and characterization of X. laevis Smac/DIABLO (XSmac/DIABLO). In this study degenerate primers based on conserved regions of human, mouse and an EST predicted Smac from X. tropicalis were used to amplify cDNA templates from X. laevis. The full length cDNA of Xenopus Smac contained a complete open reading frame of 732 bp, encoding 244 amino acids, that when expressed is observed to be approximately 27 kDa in size. The protein sequence is 49% identical and 71% similar to human Smac, and includes the motifs involved in mitochondrial targeting, and IAP-binding (AIPV). Smac expression was detected throughout early development with multiple transcripts being detected by Northern blot analysis, suggesting the presence of alternatively spliced isoforms. Exogenous expression of Xenopus Smac enhances gamma-irradiation-induced apoptosis in HeLa cells, demonstrating its functional equivalence with mammalian forms. Our study has identified the third vertebrate homologue of Smac/DIABLO, with its structural and functional similarities to mammalian Smac/DIABLO further illustrating the evolutionary conservation of apoptotic pathways across vertebrate species.

Eye and neural defects associated with loss of GDF6.

BACKGROUND: In Xenopus the bone morphogenetic protein growth and differentiation factor 6 (GDF6) is expressed at the edge of the neural plate, and within the anterior neural plate including the eye fields. Here we address the role of GDF6 in neural and eye development by morpholino knockdown experiments. RESULTS: We show that depletion of GDF6 (BMP13) resulted in a reduction in eye size, loss of laminar structure and a reduction in differentiated neural cell types within the retina. This correlated with a reduction in staining for Smad1/5/8 phosphorylation indicating a decrease in GDF6 signalling through loss of phosphorylation of these intracellular mediators of bone morphogenetic protein (BMP) signalling. In addition, the Pax6 expression domain is reduced in size at early optic vesicle stages. Neural cell adhesion molecule (NCAM) is generally reduced in intensity along the neural tube, while in the retina and brain discreet patches of NCAM expression are also lost. GDF6 knock down resulted in an increase in cell death along the neural tube and within the retina as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. CONCLUSION: Our data demonstrate that GDF6 has an important role in neural differentiation in the eye as well as within the central nervous system, and that GDF6 may act in some way to maintain cell survival within the ectoderm, during the normal waves of programmed cell death.

Gremlin - a putative pathogenic player in progressive renal disease.

Progressive renal fibrosis is the end process of renal injury leading to kidney failure. Current therapies for chronic renal failure aim to slow this process but fail to halt its progression. As the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis are unravelled, potential treatments for this growing clinical problem should emerge. Gremlin, a developmental regulator of bone morphogenetic proteins (BMPs), has recently been implicated in processes such as glomerulosclerosis, tubulointerstitial fibrosis and cellular hypertrophy, and may represent a novel therapeutic target in progressive renal diseases.

Diabetic nephropathy: renal development gone awry?

Nephrogenesis is controlled by a sequence of inductive signals between different areas of the developing kidney. As these signals are being elucidated, it has become clear that many important developmental genes are re-expressed in the mature organ following injury, possibly as part of repair and regeneration. While this reuse of developmental pathways may contribute to healing and repair, it may alternatively result in scar formation if specific components of the pathways are missing, if the temporal correlation of various elements is faulty, or if an injurious stimulus persists. In the review we will use diabetic nephropathy as an example to illustrate this paradigm in renal disease. The pathogenesis of diabetic nephropathy is complex and characterized by altered expression of many genes, including growth factors, apoptotic regulators, cellular matrix components, and cytoskeletal proteins. Many of these factors also function during kidney development. The elucidation of the roles these genes play in nephrogenesis and of their array of molecular partners and modulators may ultimately shed light on the pathogenesis of disease (and indeed vice versa), and may even suggest new therapeutic strategies.

The Xenopus pronephros as a model system for the study of kidney development and pathophysiology.

By analysing the expression and function of DN-associated genes during renal development in vivo, it may be possible to shed light on their pathogenic roles in the disease. The embryos of the African clawed frog Xenopus laevis provide a useful model for analysing early embryonic development, particularly organogenesis. Their rapid, external development and the large size of embryos allow for ease of observation and manipulation of the developmental programme. The Xenopus pronephros represents a single nephron, the basic unit of the successive vertebrate renal organs, i.e. the mesonephros and metanephros, and thus provides a useful model of nephrogenesis. Suppression subtractive hybridization was used to identify genes induced when primary cultures of mesangial cells are exposed to high extracellular glucose. Among these genes was the bone morphogenetic protein (BMP) gremlin. Interestingly gremlin is expressed in Xenopus pronephros at stage 27 where it has the potential to interact with BMPs and related regulators of nephrogenesis. Further analysis of the role of gremlin in renal development may shed light on their roles in disease.