Project description:To understand Sonic hedgehog homolog (Shh)-mediated molecular networks in the posterior second heart field (pSHF), we have employed whole genome microarray expression profiling as a discovery platform to identify genes with Shh-dependent expressional changes. We microdissected the pSHF from E9.5 embryos and compared the Shh mutant samples with than of wild-type using Agilent 4x44k Mouse Whole Genome Arrays (n = 4 WT pools and 4 Shh - /- pools). Microdissected pSHF from E9.5 mouse embryos was molecularly verified by real-time PCR. Shh mutant embryos were compared with wild-type embryos. Four independnet pools of RNAs from each biological group were measured on 1-color Agilent Mouse Whole Genome Arrays (n = 3 WT pools and 4 Shh -/- pools).
Project description:To understand Tbx5-Hedgehog molecular networks in the posterior second heart field (pSHF), we have employed whole genome microarray expression profiling as a discovery platform to identify genes with Tbx5-dependent expression changes. We microdissected the pSHF from E9.5 embryos and compared the Tbx5 mutant samples with than of wild-type using Agilent 4x44k Mouse Whole Genome Arrays (n = 4 WT pools and 4 Tbx5 -/+ pools). Microdissected pSHF from E9.5 mouse embryos was molecularly verified by real-time PCR. Tbx5 mutant embryos were compared with wild-type embryos. Four independnet pools of RNAs from each biological group were measured on 1-color Agilent Mouse Whole Genome Arrays (n = 4 WT pools and 4 Tbx5 -/+ pools).
Project description:The decline in oocyte quality is a limiting factor of female fertility; however, strategies to maintain the oocyte quality of aged women are not available. In this study, we showed that growth hormone (GH) supplementation in vivo not only alleviated the decline in oocyte number caused by aging, but also improved the quality and developmental potential of aged oocytes. Strikingly, GH supplementation reduced aneuploidy in aged oocytes. Proteomic analysis indicated that the ERK1/2 pathway was involved in the reduction in aneuploidy rate of aged oocytes, as confirmed both in vivo and in vitro. In addition, JAK2 might be involved in the regulation of ERK1/2 by GH in aged oocytes. Collectively, our findings revealed that GH supplementation protects oocytes from aging-related aneuploidy and enhances the quality of aged oocytes, and could be used to improve the outcome of assisted reproduction in aged women.
Project description:It is basically understood that male and female development is initiated by gonad differentiation into either testis or ovary. However, male embryos are reported to develop faster than female during preimplantation, implying sex differences at this stage. To learn more about when sex differentiation begins, we compared the global gene expression pattern of male and female embryos at the blastocyst stage. First, Blastocyst samples were sexed, using a novel method for non-invasive sexing of preimplantation stage mouse embryos by tagging the X chromosome with an EGFP transgene, Next, gene expression patterns of the male and female were compared using DNA microarray. ï¼³amples were collected from three independent preparations and the experiments were triplicated. Scanned microarray results were processed with Feature Extraction software (ver. 7.5, Agilent). ãThe hybridization experiments were duplicated in a reciprocal labeling manner to reduce dye integration bias, and total of six hybridizations were carried out using each 22K-1 and 22K-2 array.ã Combining plural array results and statistical analyses were carried out by Luminator software (Rosetta).
Project description:Male embryos are reported to develop faster than female in the preimplantation stage. Therefore, male and female embryos can be considered phenotypically different as early as the preimplantation stage. Employing our sexing system of enhanced green fluorescent protein (EGFP) tagging X chromosomes, we compared the global gene expression pattern of male and female embryos at the blastocyst stage using DNA microarray. Experiment Overall Design: samples were collected from three independent preparations and the experiments were triplicated. Scanned microarray results were processed with Feature Extraction software (ver. 7.5, Agilent). ãThe hybridization experiments were duplicated in a reciprocal labeling manner to reduce dye integration bias, and total of six hybridizations were carried out for the entire analysis.ã Combining plural array results and statistical analyses were carried out by Luminator software (Rosetta).
Project description:Postovulatory aging leads to the decline in oocyte quality and subsequent impairment of embryonic development, thereby reducing the success rates of assisted reproductive technology (ART). Nevertheless, potential preventative strategies to improve aging oocytes quality and the associated underlying mechanisms warrant further investigation. In this study, we identify cordycepin, an natural nucleoside analogue, as having the potential to restore the postovulatory aging-induced decline in oocyte quality, including aspects such as oocyte fragmentation, embryonic developmental competence, spindle/chromosomes morphology and mitochondrial function. Proteomic and RNA sequencing analyses revealed that cordycepin inhibited the degradation of several crucial maternal proteins and mRNAs caused by aging. Mechanistically, cordycepin was found to suppress the elevation of DCP1A protein levels by inhibiting polyadenylation during postovulatory aging, consequently impeding the decapping of maternal mRNAs. In humans, the increased degradation of DCP1A and total mRNA during aging was also inhibited by cordycepin. Collectively, our findings demonstrate that cordycepin may prevent postovulatory aging of mammalian oocytes by inhibition of maternal mRNAs degradation via DCP1A polyadenylation suppression, thereby promoting the successful rates of ART procedure.
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:Comparison of the gene expression profiles of pre-implantation embryos at day 3.5 post coitum from normal pregnant mice (control); embryos from mice treated with ICI (specific estrogen receptor inhibitor); and embryos in the oviduct that were blocked from entering the uterus by ligation. Results provide insight into the function of estrogen regulated genes and uterine factors involved in the early implantation process. RNA were extracted from 3 groups of 100-120 embryos (a) embryos at d3.5 from uterus of normal pregnant mice; (b) embryos at d3.5 from uterus of ICI treated mice; and (c) embryos at d3.5 from the ligated oviduct.
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:Transposable elements (TEs) are widely represented in eukaryotic genomes. Recently, a set of small RNAs known as rasRNAs (repeat-associated small RNAs) have been related to the down-regulation of TEs conferring a means to safeguard genome integrity. Two key members of the rasRNAs group are piRNAs and endo-siRNAs. In this study, we have performed a comparative analysis of piRNAs and endo-siRNAs present in mouse oocytes, spermatozoa and zygotes, identified by deep sequencing and bioinformatic analysis. Both piRNAs and endo-siRNAs regulate TEs in addition to other repetitive elements such as tRNAs and rRNAs, suggesting an alternative role of rasRNAs with regard to translation regulation. The detection of piRNAs and endo-siRNAs in sperm cells and revealed also in zygotes, hints to their potential delivery to oocytes during fertilization. However, a comparative assessment of the three cell types indicates that both piRNAs and endo-siRNAs are mainly maternally inherited. Finally, we have assessed the role of the different rasRNA molecules in connection with amplification processes by way of the M-bM-^@M-^\ping-pong cycleM-bM-^@M-^]. Our results suggest that the ping-pong cycle can act on other rasRNAs, such as tRNA- and rRNA-derived fragments, thus not only being restricted to TEs during gametogenesis, as was evidenced in spermatozoa, oocytes and zygotes. Comparative analysis from deep sequencing of piRNAs and endo-siRNAs in mouse oocytes, spermatozoa and zygotes