Project description:Identification of ovulation-inducing genes selected by the method for in vivo induction of oocyte maturation and ovulation in zebrafish
Project description:This project aimed at identifying developmental stage specific transcript profiles for catecholaminergic neurons in embryos and early larvae of zebrafish (Danio rerio). Catecholaminergic neurons were labeled using transgenic zebrafish strains to drive expression of GFP. At stages 24, 36, 72 and 96 hrs post fertilization, embryos were dissociated and GFP expressing cells sorted by FACS. Isolated RNAs were processed using either polyA selection and libray generation or NanoCAGE. This is the first effort to determine stage specific mRNA profiles of catecholaminergic neurons in zebrafish.
Project description:The composition of follicular fluid reflects crucial paracrine signalling between granulosa- and theca cells and the oocyte. Dynamic changes in the protein composition of human follicular fluid across multiple time points in the period where final meiotic maturation and release of the oocyte take place are previously undescribed. Twenty-five women undergoing IVF- or ICSI treatment in a standard antagonist protocol with agonist ovulation trigger were included in this prospective cohort study. From each patient one follicle was aspirated by transvaginal ultrasound guided puncture at one of five specific time points either before ovulation induction (T=0) or 12-, 17-, 32- or 36 hours after ovulation induction (five patients/time point). Proteomics was carried out by liquid chromatography-mass spectrometry. In total, 400 proteins were identified (FDR<0.05) and 30 were significantly regulated across time points (one-way ANOVA, adjusted p<0.05). Compared to compiled human plasma proteome sets, 47 proteins were unique to follicular fluid. Enriched biological functions among differentially expressed proteins included immune functions, wound healing and functions related to blood coagulation (FDR<0.02). The most profound changes occurred shortly after ovulation induction. We confirmed parallel protein expression to known granulosa cell mRNA changes and described many hitherto unknown protein expression profiles during ovulation. Thus, the study endorsed important biological functions of some proteins and suggested additional proteins, which may be crucial to intrafollicular signalling and oocyte competence that should be further investigated.
Project description:Phthalate esters (PAEs), a notable plasticizer, could be prolific contaminants in the aquatic environment, and have been shown to induce reproductive toxicity. However, studies concerning the toxicity towards aquatic species are based upon individual chemicals and the combined toxicity of PAEs to aquatic organisms remains unclear. The aim of this study was to explore the potential toxic mechanism of combined exposure to dibutyl phthalate (DBP) and diisobutyl phthalate (DiBP) in adult female zebrafish ovarian. Zebrafish were exposed to DBP, DiBP and their mixtures for 30 days, and their effects on ovarian histology, plasma sex hormones and ovarian transcriptomics were investigated. The plasma estradiol (E2) levels were significantly decreased 38.9% for DBP-1133 exposure group and 41.0% for DiBP-1038 exposure group. The percentages of late/mature oocyte were also significantly decreased 17.3% in DBP-1133 exposure and 16.2% in DiBP-1038 exposure, while those in combined exposure were not significantly affected. Nonetheless, transcriptome sequencing discovered 2564 differential expressed genes (DEGs) in zebrafish ovary after exposure to the mixtures. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified that those DEGs were involved in the neuroactive ligand-receptor interaction, GnRH, progesterone-mediated oocyte maturation, oocyte meiosis and steroid hormone biosynthesis signaling pathways. These results revealed that combined exposure showed potential reproductive toxicity at the molecular level.
Project description:Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. Late vitellogenesis: 3 samples Post-vitellogenesis: 4 samples Oocyte maturation (germinal vesicle breakdown): 6 samples
Project description:Background: Follicular growth and maturation in semi-synchronously spawning fish involve numerous cell signaling cascades and different molecular cascades are activated or inhibited during specific stages of oocyte development. The objectives of the current study were to identify molecular pathways and temporal gene expression patterns throughout a complete breeding cycle in female wild LMB to characterize the molecular sequence of events underlying follicle and ovary development. Methods: Microarray analysis was performed on eight morphologically distinct stages, from primary stages of oocyte growth to ovulation and atresia. Ovarian tissue histology, plasma vitellogenin, and sex steroids (E2 and T) were also measured to correlate molecular signaling cascades to higher levels of biological organization. Results: Global expression patterns revealed dramatic differences between early and late stages of ovarian follicle progression, with over 200 and 500 genes being differentially expressed during both ovulation and atresia respectively (p < 0.01). Time course analysis for all stages leading to ovulation and atresia identified increased expression of GABAA receptor subunits and peroxisome proliferator-activated receptor gamma during ovulation. Gene set enrichment analysis (GSEA) revealed that early stages of oocyte growth involved increases in pathways of natural killer cell and mast cell activation, as well as gap junction regulation. GSEA revealed that arachidonic acid metabolism was significantly up-regulated while CD2, fibronectin, and neuropeptides Y receptor signaling cascades were down-regulated at ovulation. Expression targets for LH signaling were decreased during vitellogenesis but increased at ovulation. GSEA revealed decreases in actin cytoskeleton regulation and receptor mediated signaling pathways involving TGF? and ephrin receptor regulation at atresia. Conclusions: This study offers new insight into the molecular pathways involved in vitellogenesis, ovulation and atresia in LMB and provides new hypotheses about the cellular pathways involved in oocyte growth and maturation. 31 microarrays on 8 unique stages of ovary development; developmental profile time course, development of LMB ovary