ABSTRACT
Follistain-like protein 1 (FSTL1), has been recently demonstrated to be involved in the embryo development of nervous system and glioblastoma. However, the role of FSTL1 in neuroinflammation remains unexplored. In this study, the expression of FSTL1 in astrocytes was verified and its role was studied in neuroinflammation induced by in vivo intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) or LPS treatment to astrocytes in vitro. FSTL1 was significantly induced after ICV LPS injection or LPS treatment. FSTL1 suppressed upregulation of pro-inflammatory cytokines in astrocytes after LPS treatment. Moreover, FSTL1 downregulated expression of pro-inflammatory cytokines through suppressing MAPK/p-ERK1/2 pathway in astrocytes. Our results suggest that FSTL1 may play an anti-inflammatory role in neuroinflammation mediated by astrocytes.
Subject(s)
Astrocytes/pathology , Cytokines/metabolism , Follistatin-Related Proteins/physiology , Inflammation/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Astrocytes/metabolism , Follistatin-Related Proteins/genetics , Follistatin-Related Proteins/pharmacology , Gene Expression Regulation , Humans , Inflammation/chemically induced , Lipopolysaccharides , MAP Kinase Signaling System/drug effects , NF-kappa B/metabolismABSTRACT
In a mammalian oocyte, completion of meiosis is suspended until fertilization by a sperm, and the cell cycle is arrested by a biochemical activity called cytostatic factor (CSF). Emi2 is one of the CSFs, and it maintains the protein level of maturation promoting factor (MPF) by inhibiting ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Degradation of Emi2 via ubiquitin-mediated proteolysis after fertilization requires phosphorylation by Polo-like kinase 1 (Plk1). Therefore, recognition and phosphorylation of Emi2 by Plk1 are crucial steps for cell cycle resumption, but the binding mode of Emi2 and Plk1 is poorly understood. Using biochemical assays and X-ray crystallography, we found that two phosphorylated threonines (Thr(152) and Thr(176)) in Emi2 are each responsible for the recruitment of one Plk1 molecule by binding to its C-terminal polo box domain (PBD). We also found that meiotic maturation and meiosis resumption via parthenogenetic activation were impaired when Emi2 interaction with Plk1-PBD was blocked by a peptidomimetic called 103-8. Because of the inherent promiscuity of kinase inhibitors, our results suggest that targeting PBD of Plk1 may be an effective strategy for the development of novel and specific contraceptive agents that block oocyte maturation and/or fertilization.
Subject(s)
Cell Cycle Proteins/chemistry , F-Box Proteins/chemistry , Peptidomimetics/chemistry , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases/chemistry , Proto-Oncogene Proteins/chemistry , Animals , Binding Sites , Cell Cycle Proteins/metabolism , Cell Differentiation/drug effects , F-Box Proteins/metabolism , Fertilization/drug effects , Meiosis/drug effects , Mesothelin , Mice , Models, Molecular , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Peptidomimetics/administration & dosage , Peptidomimetics/pharmacology , Phosphorylation , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/metabolism , Proteolysis , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/metabolism , Structure-Activity Relationship , Xenopus , Polo-Like Kinase 1ABSTRACT
Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.
Subject(s)
Actin Depolymerizing Factors/metabolism , Actins/metabolism , Asymmetric Cell Division/physiology , Oocytes/cytology , Oocytes/metabolism , Tropomyosin/metabolism , Animals , Cytoskeleton/genetics , Cytoskeleton/metabolism , Mice, Inbred ICR , Oocytes/growth & development , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering , Tropomyosin/geneticsABSTRACT
A number of germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, the role of these factors during early embryonic development has been poorly explored. In the present study, we investigated the role of SEBOX, a maternal homeobox transcription factor, during early embryonic development in porcine parthenotes. mRNA for SEBOX is preferentially expressed in oocytes, and expression persists until embryonic genome activation (EGA). Knockdown of SEBOX by siRNA disrupted early embryonic development, but not oocyte maturation. Many maternal genes essential for early embryonic development were upregulated in SEBOX-depleted embryos. Moreover, some pluripotency-associated genes, including SOX2 and NANOG, were upregulated when SEBOX was knocked down. Therefore, our data demonstrate that SEBOX is required for early embryonic development in pigs and appears to regulate the degradation of maternal transcripts and the expression of pluripotency genes.
Subject(s)
Blastocyst/metabolism , Ectogenesis , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Oocytes/metabolism , Abattoirs , Animals , Blastocyst/cytology , Cells, Cultured , Cumulus Cells/cytology , Cumulus Cells/metabolism , Embryo Culture Techniques , Embryo, Mammalian/cytology , Female , Granulosa Cells/cytology , Granulosa Cells/metabolism , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , In Vitro Oocyte Maturation Techniques , Oocytes/cytology , Parthenogenesis , RNA Interference , RNA Stability , RNA, Messenger/metabolism , RNA, Small Interfering , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Sus scrofa , Up-RegulationABSTRACT
The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.
