Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse

AbstractAlthough the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt

Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals

Genomic imprinting and parent-of-origin effects on complex traits

Parent-of-origin effects occur when the phenotypic effect of an allele depends on whether it is inherited from an individual’s mother or father. Several phenomena can cause parent-of-origin effects, with the best characterized being parent-of-origin dependent gene expression associated with genomic imprinting. Imprinting plays a critical role in a diversity of biological processes and in certain contexts it structures epigenetic relationships between DNA sequence and phenotypic variation. The development of new mapping approaches applied to the growing abundance of genomic data has demonstrated that imprinted genes can be important contributors to complex trait variation. Therefore, to understand the genetic architecture and evolution of complex traits, including complex diseases and traits of agricultural importance, it is crucial to account for these parent-of-origin effects. Here we discuss patterns of phenotypic variation associated with imprinting, evidence supporting its role in complex trait variation, and approaches for identifying its molecular signatures

Wollongong University Laboratory Microgrid: A Design for Flexibility

Microgrids are becoming increasingly important to electrical power distribution. They offer the possibility of increased reliability and lower power costs for both customers and network operators, however there are significant challenges that must be addressed before this potential can be realised. Laboratory experimentation where concepts can meet with real world complexities in a controlled environment is a crucial part of meeting these challenges. The design and implementation of a useful laboratory microgrid is a complex and expensive process and to be successful it must be done with care. This process was recently undertaken at the University of Wollongong, and the key features of the microgrid design are described within this paper along with proposed experiments to be undertaken in the near future

An Example of Freshwater Turtle Habitat Destruction by Foraging Feral Pigs in Tropical North-Eastern Australia

[Extract] Foraging by feral pigs can strongly affect vegetation assemblages and so too wider ecological processes, although how this impacts upon freshwater ecosystems and their inhabitants have seldom been studied. Between May and October 2008, we assessed the ecological effects of pig foraging activities in a pair of fenced and unfenced ephemeral floodplain lagoons at Lakefield National Park on Cape York Peninsula, and from July we estimated the comparative effect of pig disturbance on the resident freshwater turtle fauna in each lagoon. Foraging by feral pigs caused major changes to aquatic macrophyte communities and as a consequence, to the proportional amounts of open water and bare ground. The destruction of macrophyte communities and upheaved wetland sediments significantly affected wetland light penetration and water clarity, and caused prolonged anoxia and pH imbalances in the unfenced treatment. The combined effects of vegetation destruction and the subsequent excretion of pig wastes caused high levels of nutrient enrichment in the unfenced lagoon

How might an exotic fish disrupt spawning success in a sympatric native species?

Little is known of the underlying behavioural mechanisms that allow invasive species to gain an ecological foothold in novel environments. We tested how the exotic Mozambique tilapia (Oreochromis mossambicus) might affect spawning success in groups of the native Australian eastern rainbowfish (Melanotaenia splendida splendida) under experimental tank conditions using the presence of either an isolated O. mossambicus male or a small breeding group of O. mossambicus males and females. Both egg production and the proportion of eggs fertilised in M. s. splendida were significantly reduced by over 70% and 30%, respectively, in the presence of O. mossambicus breeding groups, but were not significantly affected by the presence of only a single O. mossambicus male. These results suggest that the reproductive activities of O. mossambicus may severely disrupt the spawning success of this native rainbowfish and indicates that the ecological impacts of O. mossambicus might be far more serious than is currently thought

A description of freshwater turtle habitat destruction by feral pigs in tropical north eastern Australia

Feral pigs cause major disturbance to wetland habitats that are frequented by freshwater turtles, although the effects have not been quantified. We assessed how feral pig (Sus scrofa) foraging activities affect turtle (Chelidae) habitat in a pair of fenced and unfenced ephemeral floodplain lagoons at Lakefield National Park on Cape York\ud Peninsula, Australia. Feral pigs caused major changes to aquatic macrophyte communities and as a consequence, to\ud the proportional amounts of open water and bare ground. The destruction of macrophyte communities and upheaved wetland sediments significantly affected wetland water clarity and caused prolonged anoxia and pH imbalances in the unfenced lagoon. The combined effects of vegetation destruction and the subsequent excretion of pig wastes also resulted in high nutrient enrichment. Clearly, exclusion fencing will protect freshwater turtle habitats from feral pig foraging activities, however, the choice of which lagoons to fence should only be made with an understanding of their\ud seasonal usage by freshwater turtles

Evidence that Transcription Factor AP-2γ Is Not Required for <i>Oct4</i> Repression in Mouse Blastocysts

<div><p>In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse <i>Oct4</i> promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate <i>Oct4</i> expression in the mouse TE lineage. For instance, the mouse <i>Oct4</i> distal enhancer contains an AP-2γ (Tcfap2c) binding motif that is absent in the bovine and human <i>Oct4</i> distal enhancer. Nonetheless, the functional relevance of Tcfap2c in <i>Oct4</i> repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in <i>Oct4</i> expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in <i>Oct4</i> expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on <i>Oct4</i> repression. To test whether Tcfap2c interacts with Oct4 to positively regulate <i>Oct4</i> expression, chromatin immunoprecipitation and <i>in situ</i> co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the <i>Oct4</i> distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when <i>Oct4</i> became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for <i>Oct4</i> silencing in mouse blastocysts, but may be necessary for the maintenance of <i>Oct4</i> expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of <i>Oct4</i> expression during blastocyst formation in mice.</p></div