Studies of assisted reproduction in the spotted grass frog Limnodynastes tasmaniensis: ovulation, early development and microinjection (ICSI)

Assisted Reproductive Technologies (ART) offer a wide range of techniques that have the potential to augment efforts to conserve and manage endangered amphibians and improve wild and captive population numbers. Gametes and tissues of species nearing endangered or extinct status can be cryopreserved and stored in gene banks, to provide material that can be utilised in the future as ART methods are refined. The Spotted Grass Frog, Limnodynastes tasmaniensis, is an abundant amphibian species in South-Eastern Australia of the family Myobatrachidae, that is suitable for the development of ART systems that can be applied to the threatened and endangered myobatrachid and other amphibian species native to Australia. The aim of this study was to advance the understanding of ovulation, fertilisation and embryo nic development of Lim. tasmaniensis and in vitro manipulations of reproduction and development for use in the development of advanced ART procedures such as intracytoplasmic spermatozoon injection (ICSI), androgenesis and nuclear transfer. Ovulation in amphibians can be induced by protocols utilising natural or synthetic hormones. All protocols tested on Lim. tasmaniensis in this study required two injections and the most effective protocols continued to require a first injection of pituitary extracts to induce ovulation. The second injection was, however, successfully replaced by synthetic chorionic gonadotrophin at a threshold dosage of 100 iu and halved the number of cane toads required to source the pituitaries. A combination of LHRH and Pimozide offered a less effective protocol, that did not require the use of pituitary extracts, and avoided the risk of pathogen transfer associated with unsterilised pituitary extracts. Unfertilised eggs of Lim. tasmaniensis were exposed to media of various osmolalities to determine media effects on eggs and their surrounding jelly layers that might impact on egg viability and fertilisability. Osmolality had no effect upon the egg diameter, however, rapid swelling of the jelly layers occurred within 15 minutes of exposure to various media treatments and plateaued from 30-90 minutes without further expansion. Swelling of the jelly layers was increased in hypotonic media (2.5% SAR, H2O) and minimised in the isotonic media (100% SAR). The optimal conditions for the culture of Lim. tasmaniensis eggs were identified as a holding media of 100% SAR, followed by a medium change to 2.5% SAR at insemination. This sequence of media minimised the rate of swelling of the jelly layers prior to contact with the spermatozoa, and maximised the activation of spermatozoa and eggs throughout fertilisation and embryonic development. Embryos of Lim. tasmaniensis were cultured at four temperatures (13 C, 17 C, 23 C and 29 C), to determine the effect of temperature on cleavage and embryonic development rates. Embryonic development progressed through a sequence of stages that were not altered by changes in temperature. However cleavage rates were affected by changes in temperature as compared with normal embryonic growth at 23 C. Embryonic development was suspended at the lowest temperature (13 C) while embryonic viability was maintained. A moderate decrease in temperature (17 C) slowed cleavage, while the highest temperature (29 C) increased the cleavage rate, but decreased the embryo survival. Rates of embryonic development can be manipulated by changes in temperature and this method can be used to source blastomeres of a specific size/stage at a predetermined age or halt cleavage at specific stages for embryos or embryo derived cells to be included in ART procedures. This study produced the first report of the application of Intracytoplasmic Spermatozoon Injection (ICSI) in an Australian amphibian. Eggs that were activated by microinjection with a single spermatozoon (n=50) formed more deep, but abnormal, cleavage furrows post-injection (18/50, 36%), than surface changes (12/50, 24%). This result is in contrast to eggs injected without a spermatozoon (n=42), where the majority of eggs displayed limited surface changes (36/42, 86%), and few deep, abnormal furrows (3/42, 7%). Three advanced embryos (3/50, 6%) were produced by ICSI that developed to various stages within the culture system. Technical difficulties were encountered that prevented the generation of any metamorphs from ICSI tadpoles. Nevertheless, when these blocks to ICSI are overcome, the ICSI procedure will be both directly useful as an ART procedure in its own right, and the associated refinement of micromanipulation procedures will assist in the development of other ART procedures in Lim. tasmaniensis. A greater understanding of basic reproductive and developmental biology in Lim. tasmaniensis would greatly facilitate refinement of fertilisation by ICSI. Assisted Reproductive Technologies, in conjunction with gene banks may in the future regenerate extinct amphibian species, and assist in the recovery of declining amphibian populations nationally and worldwide.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) slows cancer development but increases metastases in TRAMP prostate cancer prone mice

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1fms) to produce syngeneic TRAMPfmsmic-1 mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1fms and syngeneic C57BL/6 mice. Whilst TRAMPfmsmic-1 survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1fms mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

The cancer stem-cell hypothesis : its emerging role in lung cancer biology and its relevance for future therapy

The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review.

Experimental and modelling investigation of monolayer development with clustering

Standard differential equation–based models of collective cell behaviour, such as the logistic growth model, invoke a mean–field assumption which is equivalent to assuming that individuals within the population interact with each other in proportion to the average population density. Implementing such assumptions implies that the dynamics of the system are unaffected by spatial structure, such as the formation of patches or clusters within the population. Recent theoretical developments have introduced a class of models, known as moment dynamics models, which aim to account for the dynamics of individuals, pairs of individuals, triplets of individuals and so on. Such models enable us to describe the dynamics of populations with clustering, however, little progress has been made with regard to applying moment dynamics models to experimental data. Here, we report new experimental results describing the formation of a monolayer of cells using two different cell types: 3T3 fibroblast cells and MDA MB 231 breast cancer cells. Our analysis indicates that the 3T3 fibroblast cells are relatively motile and we observe that the 3T3 fibroblast monolayer forms without clustering. Alternatively, the MDA MB 231 cells are less motile and we observe that the MDA MB 231 monolayer formation is associated with significant clustering. We calibrate a moment dynamics model and a standard mean–field model to both data sets. Our results indicate that the mean–field and moment dynamics models provide similar descriptions of the 3T3 fibroblast monolayer formation whereas these two models give very different predictions for the MDA MD 231 monolayer formation. These outcomes indicate that standard mean–field models of collective cell behaviour are not always appropriate and that care ought to be exercised when implementing such a model.

Increased plant volatile production affects oviposition, but not larval development, in the moth Helicoverpa armigera

It is well established that herbivorous insects respond to changes in plant odour production, but little attention has been given to whether these responses relate to direct fitness costs of plant volatile production on insect growth and survival. Here, we use transgenic Nicotiana tabacum (tobacco) plants that produce relatively large amounts of the volatile (S)-linalool to study whether the responses of egg-laying herbivorous insects to linalool production relate directly to the growth and survival of offspring. In choice tests, fewer eggs were laid on transgenic plants compared with non-transformed controls, indicating that increased linalool emissions have a deterrent effect on Helicoverpa armigera oviposition. Larval survival and larval mass after feeding on transgenic leaves, however, was comparable to non-transformed controls. (S)-linalool, whether in volatile or sequestered form, does not appear to have a direct effect on offspring fitness in this moth. We discuss how the ecology of this polyphagous moth species may necessitate a high tolerance for certain volatiles and their related non-volatile compounds, and suggest that responses by adult female H. armigera moths towards increased linalool production may be context specific and relate to other indirect effects on fitness.

Metabolomics coupled with proteomics advancing drug discovery toward more agile development of targeted combination therapies

To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations of plant species and/or minerals, called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics, and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of hepatic injury. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect, and activates both intrinsic and extrinsic pathways.

Development of an eight gene expression profile implicating human breast tumours of all grade

The goal of improving systemic treatment of breast cancers is to evolve from treating every patient with non-specific cytotoxic chemotherapy/hormonal therapy, to a more individually-tailored direct treatment. Although anatomic staging and histological grade are important prognostic factors, they often fail to predict the clinical course of this disease. This study aimed to develop a gene expression profile associated with breast cancers of differing grades. We extracted mRNA from FFPE archival breast IDC tissue samples (Grades I–III), including benign tumours. Affymetrix GeneChip� Human Genome U133 Plus 2.0 Arrays were used to determine gene expression profiles and validated by Q-PCR. IHC was used to detect the AXIN2 protein in all tissues. From the array data, an independent group t-test revealed that 178 genes were significantly (P B 0.01) differentially expressed between three grades of malignant breast tumours when compared to benign tissues. From these results, eight genes were significantly differentially expressed in more than one comparison group and are involved in processes implicated in breast cancer development and/or progression. The two most implicated candidates genes were CLD10 and ESPTI1 as their gene expression profile from the microarray analysis was replicated in Q-PCR analyses of the original tumour samples as well as in an extended population. The IHC revealed a significant association between AXIN2 protein expression and ER status. It is readily acknowledged and established that significant differences exist in gene expression between different cancer grades. Expansion of this approach may lead to an improved ability to discriminate between cancer grade and other pathological factors.

Expression of Caenorhabditis elegans RNA-directed RNA polymerase in transgenic Drosophila melanogaster does not affect morphological development

Drosophila melanogaster, along with all insects and the vertebrates, lacks an RdRp gene. We created transgenic strains of Drosophila melanogaster in which the rrf-1 or ego-1 RdRp genes from C. elegans were placed under the control of the yeast GAL4 upstream activation sequence. Activation of the gene was performed by crossing these lines to flies carrying the GAL4 transgene under the control of various Drosophila enhancers. RT-PCR confirmed the successful expression of each RdRp gene. The resulting phenotypes indicated that introduction of the RdRp genes had no effect on D. melanogaster morphological development. © 2010 Springer Science+Business Media B.V.

Targeting amino acid transport in metastatic castration-resistant prostate cancer : effects on cell cycle, cell growth, and tumor development

Background L-type amino acid transporters (LATs) uptake neutral amino acids including L-leucine into cells, stimulating mammalian target of rapamycin complex 1 signaling and protein synthesis. LAT1 and LAT3 are overexpressed at different stages of prostate cancer, and they are responsible for increasing nutrients and stimulating cell growth. Methods We examined LAT3 protein expression in human prostate cancer tissue microarrays. LAT function was inhibited using a leucine analog (BCH) in androgen-dependent and -independent environments, with gene expression analyzed by microarray. A PC-3 xenograft mouse model was used to study the effects of inhibiting LAT1 and LAT3 expression. Results were analyzed with the Mann-Whitney U or Fisher exact tests. All statistical tests were two-sided. Results LAT3 protein was expressed at all stages of prostate cancer, with a statistically significant decrease in expression after 4–7 months of neoadjuvant hormone therapy (4–7 month mean = 1.571; 95% confidence interval = 1.155 to 1.987 vs 0 month = 2.098; 95% confidence interval = 1.962 to 2.235; P = .0187). Inhibition of LAT function led to activating transcription factor 4–mediated upregulation of amino acid transporters including ASCT1, ASCT2, and 4F2hc, all of which were also regulated via the androgen receptor. LAT inhibition suppressed M-phase cell cycle genes regulated by E2F family transcription factors including critical castration-resistant prostate cancer regulatory genes UBE2C, CDC20, and CDK1. In silico analysis of BCH-downregulated genes showed that 90.9% are statistically significantly upregulated in metastatic castration-resistant prostate cancer. Finally, LAT1 or LAT3 knockdown in xenografts inhibited tumor growth, cell cycle progression, and spontaneous metastasis in vivo. Conclusion Inhibition of LAT transporters may provide a novel therapeutic target in metastatic castration-resistant prostate cancer, via suppression of mammalian target of rapamycin complex 1 activity and M-phase cell cycle genes.

Identification and characterization of flowering genes in kiwifruit : sequence conservation and role in kiwifruit flower development

Background Flower development in kiwifruit (Actinidia spp.) is initiated in the first growing season, when undifferentiated primordia are established in latent shoot buds. These primordia can differentiate into flowers in the second growing season, after the winter dormancy period and upon accumulation of adequate winter chilling. Kiwifruit is an important horticultural crop, yet little is known about the molecular regulation of flower development. Results To study kiwifruit flower development, nine MADS-box genes were identified and functionally characterized. Protein sequence alignment, phenotypes obtained upon overexpression in Arabidopsis and expression patterns suggest that the identified genes are required for floral meristem and floral organ specification. Their role during budbreak and flower development was studied. A spontaneous kiwifruit mutant was utilized to correlate the extended expression domains of these flowering genes with abnormal floral development. Conclusions This study provides a description of flower development in kiwifruit at the molecular level. It has identified markers for flower development, and candidates for manipulation of kiwifruit growth, phase change and time of flowering. The expression in normal and aberrant flowers provided a model for kiwifruit flower development.

The biology of breast tumor progression : acquisition of hormone independence and resistance to cytotoxic drugs

Many breast tumors appear to follow a predictable clinical pattern, being initially responsive to endocrine therapy and to cytotoxic chemotherapy but ultimately exhibiting a phenotype resistant to both modalities. Using the MCF-7 human breast cancer cell line as an example of an 'early' phenotype (estrogen and progesterone receptor positive, steroid responsive, low metastatic potential), we have isolated and characterized a series of hormone-independent but hormone-responsive variants (MIII and MCF7/LCC1). However, these variants remain responsive to both antiestrogens and cytotoxic drugs (methotrexate and colchicine). MIII and MCF7/LCCl cells appear to mimic some of the critical aspects of the early progression to a more aggressive phenotype. An examination of the phenotype of these cells suggests that some hormone-independent breast cancer cells are derived from hormone-dependent parental cells. The development of a hormone-independent phenotype can arise independently of acquisition of a cytotoxic drug resistant phenotype.

The epithelial-mesenchymal transition : new insights in signaling, development, and disease

The conversion of an epithelial cell to a mesenchymal cell is critical to metazoan embryogenesis and a de. ning structural feature of organ development. Current interest in this process, which is described as an epithelial- mesenchymal transition (EMT), stems from its developmental importance and its involvement in several adult pathologies. Interest and research in EMT are currently at a high level, as seen by the attendance at the recent EMT meeting in Vancouver, Canada (October 1-3, 2005). The meeting, which was hosted by The EMT International Association, was the second international EMT meeting, the . rst being held in Port Douglas, Queensland, Australia in October 2003. The EMT International Association was formed in 2002 to provide an international body for those interested in EMT and the reverse process, mesenchymal-epithelial transition, and, most importantly, to bring together those working on EMT in development, cancer, . brosis, and pathology. These themes continued during the recent meeting in Vancouver. Discussion at the Vancouver meeting spanned several areas of research, including signaling pathway activation of EMT and the transcription factors and gene targets involved. Also covered in detail was the basic cell biology of EMT and its role in cancer and . brosis, as well as the identi. cation of new markers to facilitate the observation of EMT in vivo. This is particularly important because the potential contribution of EMT during neoplasia is the subject of vigorous scientific debate (Tarin, D., E.W. Thompson, and D.F. Newgreen. 2005. Cancer Res. 65:5996-6000; Thompson, E.W., D.F. Newgreen, and D. Tarin. 2005. Cancer Res. 65:5991-5995).

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma

Cancer can be defined as a deregulation or hyperactivity in the ongoing network of intracellular and extracellular signaling events. Reverse phase protein microarray technology may offer a new opportunity to measure and profile these signaling pathways, providing data on post-translational phosphorylation events not obtainable by gene microarray analysis. Treatment of ovarian epithelial carcinoma almost always takes place in a metastatic setting since unfortunately the disease is often not detected until later stages. Thus, in addition to elucidation of the molecular network within a tumor specimen, critical questions are to what extent do signaling changes occur upon metastasis and are there common pathway elements that arise in the metastatic microenvironment. For individualized combinatorial therapy, ideal therapeutic selection based on proteomic mapping of phosphorylation end points may require evaluation of the patient's metastatic tissue. Extending these findings to the bedside will require the development of optimized protocols and reference standards. We have developed a reference standard based on a mixture of phosphorylated peptides to begin to address this challenge.

B-Raf mutation : a key player in molecular biology of cancer

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common.