Project description:A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Since the signaling molecules RAS and ERK1/2 are activated by a LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBP is a critical downstream mediator of ERK1/2 activation. These mouse models provide in vivo systems in which to define the context specific and molecular mechanisms by which granulosa cells respond to LH and these mechanisms are relevant to the regulation of human fertility and infertility.
Project description:Peripheral light harvesting (LH) antenna complexes have been studied extensively in the purple nonsulfur bacterium Rhodopseudomonas palustris because it produces different types of LH complexes under high light intensities (LH2 complex) and low light intensities (LH3 and LH4 complex). The ability of R. palustris to alter its peripheral LH complexes in response to changes in light intensity is attributed to the multiple operons that encode the a and b peptides that make up these complexes, whose expression is affected by light intensity, light quality, and oxygen tension. However, low resolution structures, amino acid similarities between the complexes, and a lack of transcriptional analysis made it difficult to determine the LH complexes composition and functions under different light intensities. It was also unclear how much diversity of the R. palustris LH complexes exists in nature.Results: To gain insight into the composition of the LH complexes, their function under high light intensities and low light intensities, and their prevalence in the environment we undertook an integrative genomics approach using 15 closely related R. palustris strains isolated from the environment and 5 R. palustris ecotypes whose genomes have been sequenced. We sequenced the genomes for the 15 closely related strains and using RNA-seq carried out transcriptomic analysis on all 20 strains grown under high light intensity and low light intensity. We were able to determine that even closely related R. palustris strains had differences in their pucBA gene content and expression, even under the same growth conditions. We also found that the LH2 complex could compensate for the lack of an LH4 complex under LL intensities but not under extremely LL intensities. Conclusions: This is the first time an integrative genomics approach has been used to study light harvesting in the environment. The variation observed in LH gene composition and expression in environmental isolates of R. palustris likely reflects how these strains have adapted to specific light conditions in the environment. We have also shown that there is redundancy between some of the LH complexes under certain light intensities, which may partially explain why multiple operons encoding LH complexes have evolved and been maintained in R. palustris. Examing the variation observed in LH gene composition and expression in various environmental isolates
Project description:Competent endometrial receptivity is a prerequisite for successful embryo implantation. Identification of novel key molecules involved in endometrial receptivity is essential to better interpret human implantation and improve pregnancy rates in assisted reproduction treatment. The isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to profile the proteomes of the prereceptive( LH+2, n=4) and receptive (LH+7, n=4) endometrial tissues. One hundred seventy-three differentially expressed proteins (DEPs) between LH+2 and LH+7 endometrial samples were identified. Integrated analysis of the proteomic data and published transcriptomic data was performed to identify the concordant DEPs with differential expression at both mRNA and protein levels. The protein-protein-interaction (PPI) network analysis were performed on concordant DEPs.We first identified 63 novel concordant DEPs and 5 hub proteins (ACSL4, ACSL5, COL1A1, PTGS1 and PLA2G4F) between LH+2 and LH+7 endometrial samples. ACSL4 was predominantly expressed in endometrial epithelial cells and its expression was significantly upregulated in the LH+7 endometrium and significantly downregulated in repeated implantation failure (RIF) patients. Knockdown of ACSL4 in endometrial epithelial cells induced the down-regulation of endometrial receptivity markers (HOXA10, COX2 and LIF) and the significant decrease of implantation rate during in vitro implantation analysis. This study provides the first gel-independent quantitative proteomes of the LH+2 and LH+7 human endometrium using iTRAQ technology. The identified concordant DEPs and hub proteins open a new avenue for future studies aimed at elucidating the underlying mechanisms governing endometrial receptivity. ACSL4 was identified as a novel regulatory molecule in the establishment of endometrial receptivity and might play important roles during implantation.
Project description:A surge of luteinizing hormone (LH) from the pituitary gland triggers ovulation, oocyte maturation, and luteinization for successful reproduction in mammals. Since the signaling molecules RAS and ERK1/2 are activated by a LH surge in granulosa cells of preovulatory follicles, we disrupted Erk1/2 in mouse granulosa cells and provide in vivo evidence that these kinases are necessary for LH-induced oocyte resumption of meiosis, ovulation, and luteinization. In addition, biochemical analyses and selected disruption of the Cebpb gene in granulosa cells demonstrate that C/EBP is a critical downstream mediator of ERK1/2 activation. These mouse models provide in vivo systems in which to define the context specific and molecular mechanisms by which granulosa cells respond to LH and these mechanisms are relevant to the regulation of human fertility and infertility. Immature wild type or ERK1/2 conditonal knock-out mice were injected with 5IU equine chorionic gonadotropin (eCG)-48h followed by 5 IU hCG injection. The ovarian granulosa cells were collected at hCG 0h, 2.5h, or 4h and the gene expression pforiles were compared by microarray method.
Project description:Granulosa cells from three different stages were used to assess the short- and long-term effects of luteinizing hormone (LH) on follicle differentiation: 1) 2 h before induction of the LH surge, 2) 6 h and 3) 22 h after the LH surge. Three time points experiment: 2h pre-LH, 6h post-LH and 22h post-LH. Granulosa cells from the 6h post-LH and 22h post-LH were compared to the 2h pre-LH. Biological replicates: 4 from each time point. One replicate per array. Dye-swaps were performed.
Project description:In this study, we took advantage of deep sequencing approaches to investigate the miRNA expression profiles of human endometrium on days LH+2 and LH+7 in natural cycles, and compare them with those on days hCG+4 and hCG+7 in stimulated cycles during IVF treatment.