Expression data from bovine corpus luteum treated with equine chorionic gonadotropin (eCG)
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ABSTRACT: In order to indentify genes regulated by eCG, and involved in CL development and progesterone increases, the transcriptome was evaluated using the microarray technology Cows (Bos indicus) were divided into control, stimulated and superovulated groups. The stimulated group received 400 IU of eCG on day 8 and the superovulated group received 2000 IU of eCG on day 4 after the beginning of synchronization. Corpus luteum were collected at day 6 after ovulation e the trasncripitome was available by microarray.
Project description:Cumulus-oocyte complexes were isolated at seperate time-points to generate temporal complexes. Targets from two biological replicates at each time point (0h, 8h, 16h post-hCG treatment) were generated and the expression profiles were determined using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Comparisons between the sample groups allow the identification of genes with temporal expression patterns. Experiment Overall Design: 2 eCG-primed (48h) pooled cumulus-oocyte complexes, 2 eCG-primed (48h) hCG-treated (8h) pooled cumulus-oocyte complexes, and 2 eCG-primed (48h) hCG-treated (16h) pooled cumulus-oocyte complexes replicates were analyzed
Project description:Gene regulation at the maternal-embryonic transition in the pre-implantation mouse embryo is not well understood. We knock down Ccna2 to establish proof-of-concept that antisense morpholino oligonucleotides can be used to target specific genes. We applied this strategy to study Oct4 and discovered that Oct4 is required prior to blastocyst development. Specifically, gene expression is altered as early as the 2-cell stage in Oct4-knockdown embryos. Distinct subsets of genes are differentially expressed between Oct4 and Ccna2-knockdown embryos, and indicated differential functions. Further, a large panel of genes were confirmed to be differentially-expressed in Oct4-knockdown embryos by quantitative, real time RT-PCR. Keywords: gene knockdown 3-5 week old wild type F1 (C57BL6xDBA/2) females (Charles River) were superovulated by intraperitonial injections of 5 IU of pregnant mare’s serum gonadotropin (Sigma) followed by 5 IU of human chorionic gonadotropin (Sigma) 48 hours later, and mated overnight with wild type males. Mice were sacrificed by cervical dislocation 17 hours after hCG injection, and 1-cell embryos were released from oviducts. Cumulus cells were removed by hyaluronidase (Sigma) treatment and pipetting. Pre-implantation embryos at the two pronuclei stage were recovered, pooled from 3-6 females in M2 media (Chemicon International), followed by immediate cytoplasmic microinjection of gene-specific antisense morpholino oligonucleotides and culture in Human Tubal Fluid with 10% serum supplement (In-Vitro Fertilization, Inc.) microdrops under mineral oil (Sigma) in mixed gas (90% nitrogen, 5% oxygen, 5% carbon dioxide; Praxair) at 37°C, and cultured at ten embryos per 20 μL drop. Uninjected control embryos derived from the same embryo pool, and were placed in identical conditions in parallel, except that they were not injected.
Project description:WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine if it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1-/- females were sterile due to uterine fibrosis and ovarian tubulostromal hyperplasia, the majority were subfertile, producing M-bM-^IM-^H1 less pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1-/- mice had normal weights, numbers of follicles, steroid hormone production and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1-/- mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4tm1.1Boer/tm1.1Boer;Tg(CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1-/- COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but is unlikely to function as the sole ovarian WNT4 receptor. Triplicate RNA samples from granulosa cells of Fzd1 KO mice are compared to triplicate RNA samples from granulosa cells of control Fzd1 WT mice
Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. We use mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. In this experiment, we are testing how different GC transcriptional profiles correlate with embryo quality. 3M old C57BL6/J mice were naturally or superovulated and COCs singularized as described above. Granulosa cells were immediately flash frozen and matched oocytes were divided into individual drops of CARD media under mineral oil for fertilization. The oocytes were incubated in 37oC, 5% CO2 incubator for 30-40 min prior to fertilization. Frozen Mus musculus (C57BL6/J) male sperm straws were thawed and capacitated in Fertiup media for 30 min. Fertilization was achieved by combining 2.5 µl of activated sperm with 20 µl CARD media drop containing a single oocyte and incubated at 37oC, 5% CO2 for 3 hours. Then, oocytes were individually washed in Human Tubal Fluid (HTF) media drops and transferred to 20 µl HFT drops for overnight culture. After 24 hours, fertilization rate was evaluated by 2-cell stage and embryos transferred to G1+ media for further development. Early morula stage embryos were continuously collected based on observation 62-64 h post-fertilization. For late blastocysts, fertilization media was additionally changed to G2+ after 48h post- fertilization and blastocysts collected at 108h post-fertilization. For collection, the embryos were washed in DPBS and flash frozen. Granulosa cells and embryo samples were further processed by Smart-seq2, the cDNA amplification PCR cycle number for embryos was 16-17.
Project description:Background: The possible impact of changes in diet composition for the intestinal microbiome is mostly studied after some days of adaptation to the diet of interest. The question arises if few days are enough to reflect the microbial response to the diet by changing the community composition and function. The present study investigated the fecal microbiome of pigs in a time span of four weeks after a dietary change to get an insight of the needed adaptation period. Four different diets were used differing in either protein source (field peas meal vs. soybean meal) or the concentration of calcium and phosphorus (CaP). Results: Twelve pigs were sampled at seven time points within four weeks after the dietary change. Fecal samples were used to sequence the 16S rDNA amplicons, to analyse the microbial proteins via LC-MS/MS and to determine the SCFA production. The analysis of OTU abundances and quantification values of proteins showed a significant separation of three periods of time (p=0.001). Samples from the first day are used to define the ‘Zero phase’, samples of weeks one and two are combined as ‘metabolic phase’ and an ‘equilibrium phase’ was defined based on samples from week three and four. Only in this last phase, a separation according to the supplementation of CaP was significantly detectable (p=0.001). No changes were found based on the corn-soybean meal or corn-field peas administration. The analysis of possible factors causing this significant separation showed only an overall change of bacterial members and functional properties. The metaproteomic approach yields a total of about 9700 proteins, which were used to deduce possible metabolic functions of the bacterialcommunity.
Project description:Identification of protein changes and pathways involved in combined effects of aflatoxin B1 and ochratoxin A and the preventive effect of dietary by-product antioxidants administration against these mycotoxins damage.
Project description:In order to establish an obese mouse model, female mice were continuously fed with a high-fat diet (HFD) or a normal diet (control) for 16 weeks beginning at three weeks of age. In this paper, these mice are termed ‘HFD mice’ and ‘control mice’, respectively. Accordingly, we call their oocytes ‘HFD oocytes’ and ‘control oocytes’. Substantial evidence indicates that the effects of maternal obesity on embryo/offspring development can be attributed to factors within the oocyte (9). To identify such potential effectors, we performed a comparative proteomic analysis of ovulated MII oocytes from control and HFD mice.
Project description:Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. To get insights of the repertoire of MB genes that control initiation and maintenance of neural differentiation as well as the repertoire of neural factors that may have functions in the synaptic plasticity of MB neurons during learning and memory, we compared the transcript profiles between wild type and MB-ablated brains using a Drosophila whole-genome microarray. Newly hatched larvae were briefly administered with a DNA-synthesis inhibitor, hydroxyurea, and raised to adults, from which total brain RNA was analyzed. Experiment Overall Design: Two conditions analyzed: Control Brains and Musroom Body-ablated brains. Experiment Overall Design: Each condition was analyzed in triplicate.
Project description:To study the physiological role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had significantly reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that, while Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty, they had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RTPCR analyses of Wnt4flox/-;Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1 and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 also regulates a series of genes involved in late follicle development and the cellular stress response via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development, and may act by regulating granulosa cell functions including steroidogenesis. Experiment Overall Design: Granulosa cells were obtained from 20-26 day-old mice of various genotypes 48h after eCG treatment, using the needle puncture method as previously described. Cells were then infected with adenoviruses to express eGFP or Cre, or overexpress WNT4 in serum-free medium, and subsequently harvested for RNA extraction as described below. Preliminary experiments demonstrated that an infection efficiency of >80% could be obtained at an MOI of ~50 (as determined by analysis of fluorescent signal in Ad-eGFP-infected cells) and that the Ad-Cre and Ad-WNT4 viruses produced efficient recombination of the floxed Ctnnb1 alleles and robust WNT4 overexpression, respectively. Microarray analyses were done using triplicate RNA samples from each adenoviral treatment described above, and using mouse expression set 430 microarrays (Affymetrix, Santa Clara, CA).
Project description:Dysregulation of adenosine (Ado) homeostasis has been observed in both rodent models and human patients of Huntington’s disease (HD). However, the underlying mechanisms of Ado signaling in HD pathogenesis is still unclear. In this study we examined influence of Ado signaling on Drosophila HD model. We further examined the transcription profile of AdoR mutants by microarray analysis to identified a downstream target of AdoR signaling, which mediates the AdoR effects on HD pathology. Our findings have important implications for the crosstalk between Ado signaling and pathogenic effects of HD as well as other human diseases associated with polyglutamine aggregation.