Transcriptome assay of materal mRNA degradation in Btg4-deleted oocytes
ABSTRACT: We analyzed the functions of BTG family proteins in maternal mRNA degradation in mouse oocytes. By comparing the degradation of transcripts in WT oocytes and KO oocytes, we are able to know the defects in maternal mRNA clearance in BTG4-deleted oocytes, and identified the BTG4 target genes in oocyte cyplasmic maturation. 2 WT oocyte samples at GV stage, 2 WT oocyte samples at MII stage, 2 Btg4-/- oocyte samples at GV stage and 2 Btg4-/- oocyte samples at MII stage?2 WT embryo samples at zygote stage, 2 WT embryo samples at 2-cell stage, 2 Btg4-/- embryo samples at zygote stage and 2 Btg4-/- embryo samples at 2-cell stage , and a WT GV oocyte, a WT MII oocyte, a Erk-/- GV oocyte and a Erk-/- MII oocyte are performed RNA sequencing.
Project description:There is massive destruction of transcripts during maturation of mouse oocytes. The objective of this project was to identify and characterize the transcripts that are degraded versus those that are stable during the transcriptionally silent germinal vesicle (GV)-stage to metaphase II (MII)-stage transition using the microarray approach. A system for oocyte transcript amplification using both internal and 3'-poly(A) priming was utilized to minimize the impact of complex variations in transcript polyadenylation prevalent during this transition. Transcripts were identified and quantified using Affymetrix Mouse Genome 430 v2.0 GeneChip. The significantly changed and stable transcripts were analyzed using Ingenuity Pathways Analysis and GenMAPP/MAPPFinder to characterize the biological themes underlying global changes in oocyte transcripts during maturation. It was concluded that the destruction of transcripts during the GV to MII transition is a selective rather than promiscuous process in mouse oocytes. In general, transcripts involved in processes that are associated with meiotic arrest at the GV-stage and the progression of oocyte maturation, such as oxidative phosphorylation, energy production, and protein synthesis and metabolism, were dramatically degraded. In contrast, transcripts encoding participants in signaling pathways essential for maintaining the unique characteristics of the MII-arrested oocyte, such as those involved in protein kinase pathways, were the most prominent among those stables. Experiment Overall Design: Comparison immature GV-stage oocyte (3 biological replicates) with mature MII-stage oocytes (3 biological replicates)
Project description:We analyzed the functions of ERK in maternal mRNA degradation in mouse oocytes. By comparing the degradation of transcripts in WT oocytes and KO oocytes, we are able to know the defects in maternal mRNA clearance in ERK-deleted oocytes, and identified the ERK target genes in oocyte maturation. Overall design: a WT GV oocyte, a WT MII oocyte, a Erk-/- GV oocyte and a Erk-/- MII oocyte are performed RNA sequencing.
Project description:In cattle, almost all fully grown vesicle stage oocytes (GV) have the ability to resume meisos, develop to Metaphase II stage (MII), support fertilization and progress through the early embryonic cycles in vitro. Yet without intensive selection, the majority fail to develop to the blastocyst stage. Using the Affymetrix Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of the bovine oocyte and to identify the key pathways associated with oocyte meiotic maturation and developmental potential. Immature and in vitro matured bovine oocytes were collected for RNA extraction and hybridization on Affymetrix GeneChip Bovine Genome array. Careful removal of cumulus and selection of oocytes was carried out under the stereo microscope in order to examine the actual cumulus-free temporal oocyte gene expression profiles. Immature oocytes at time 0 h and in vitro matured oocytes at 24 h were collected for analysis.
Project description:We generated maternal and paternal mouse models with Yap1-deletion, and elucidated the function of maternal YAP in zygotic genome activation. RNA sequencing with 2 WT oocyte samples at GV stage, 2 YAP-/- oocyte samples at GV stage, 1 WT embryo samples at 4cell stage, 1 YAP-/- embryo samples at at 4-cell stage.
Project description:The oocyte maturation is a poorly understood process. Patl2 is involved in human bad egg syndrome and is a translation factor. The aim of this study was to assess the impact of the lack of Patl2 on the transcriptomes of GV and MII oocytes. Overall design: Mouse oocytes from wild type and Patl2-knock-out mouse were collected at GV and MII stages. Total RNA were purified and analyzed on mouse ClariomD Affymetrix arrays. The experiment included five GV stage oocyte samples (two wild type and three Patl2-KO) and five GMII stage oocytes samples (two wild-type and three Patl2-KO).
Project description:Expression data from prepubertal, peripubertal, and adult derived mouse oocytes, and from germinal vesicle (GV), in vivo matured, and in vitro matured mouse oocytes. Oocytes derived from prepubertal females, or oocytes matured in vitro, are less developmentally competent compared to adult derived, or in vivo matured, oocytes, indicated by decreased embryonic development. One potential mechanism for decreased developmental potetential in prepubertal or in vitro matured oocytes is inadequate or inappropriate RNA degradation during oocyte maturation (progression from GV to MII). To investigate mechanisms involved in establishing oocyte cytoplasmic maturation and developmental competence, Affymetrix GeneChip microarrays were used. Keywords: Oocyte developmental competence The study encompassed three experimental designs using female B6D2F1 mice: 1) In vitro matured oocytes were obtained from d20 (prepubertal), d26 (peripubertal), and 7-8 wk old (adult) mice; 2) in vivo and in vitro matured oocytes were obtained from d26 mice; and 3) GV, in vivo matured, and in vitro matured oocytes were obtained from 7-8 wk old mice. RNA was extracted from pools of 150 oocytes and hybridized onto the Affymetrix microarrays.
Project description:Study Question: What effects do maternal age and oocyte maturation stage have on the human oocyte transcriptome that may be associated with oocyte developmental potential? Summary Answer: Although polyadenylated transcript abundance changes during human oocyte maturation irrespective of age, young (YNG) and advanced maternal age (AMA) metaphase II (MII) oocytes exhibit divergent transcriptomes. What is known already: Maternal age and maturation stage affect oocyte polyadenylated transcript abundance in human oocytes. Although RNA-Seq analysis of single human MII oocytes has been conducted, comparison of the germinal vesicle (GV) and MII oocyte transcriptomes has not been investigated using RNA-Seq, a technique that could provide novel insight into oocyte maturation and age-associated aberrations in gene expression. Participants / materials, settings, methods: Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided egg retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 hour incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads in HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, Weighted Gene Correlation Network Analysis (WGCNA), 3’UTR motif analysis, Ingenuity Pathway Analysis) were performed. Main results and the role of chance: Within the 12,770 expressed genes in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least 3 of 5 replicates for a minimum of one sample type), 458 and 3,506 genes significantly (adjusted p < 0.05 and log2 fold change > 1) increased and decreased in polyadenylated transcript abundance during oocyte maturation, respectively. The differentially expressed genes were enriched (FDR < 0.05) for biological functions and canonical pathways related to cell cycle and mitochondrial function. The majority (76%) and minority (25%) of up- and down-regulated transcripts with a complete 3’UTR were predicted to be targets of cytoplasmic polyadenylation (910 total genes), respectively. Differential gene expression analysis between young and advanced maternal age oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted p < 0.1 and log2 fold change > 1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5, and PRDX1, which have been reported to affect oocyte developmental potential and be markers of oocyte quality. Despite similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle, and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were determined to be correlated (p < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation. Limitations, reasons for caution: The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by in vitro maturation of patient oocytes, which has the benefit of identifying intrinsic differences between samples, but may not be completely representative of in vivo matured oocytes. Thus, these factors should be considered when interpreting the results. Wider Implications of the findings: Transcriptome profiles of young and advanced maternal age oocytes, particularly at the MII stage, suggest aberrant transcript abundance contributes to the age-associated decline in fertility. Overall design: Study design, size, and duration: The effect of oocyte maturation (cross-sectional analysis) and age (longitudinal analysis) on polyadenylated transcript abundance were analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (<30 years; average age 26.8; range 20-29) and five advanced maternal age (≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 young (5 GV and 5 MII) and 10 advanced maternal age (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis.
Project description:The study tests the hypothesis that maternal mRNA translation in oocytes is sensitive to the environment in which the oocytes mature. Amphiregulin (AREG) is a critical signal for oocyte maturation but also for oocyte developmental competence. Here we have used a genome-wide approach to determine whether the oocyte translational program is affected when oocytes mature in vivo in the absence of AREG. To this aim, polysome arrays were used to define patterns of transcript recruitment to the polysomes in oocytes derived from wild type mice and mice homozygous null for the Areg gene. Forty-eight hours (h) after PMSG injection, mice were stimulated with hCG for 0, or 14 h, and GV, and MII stage oocytes were collected. Polysome bound mRNAs were purified, reverse-transcribed and linearly amplified with WT-Ovation FFPE RNA Amplification System V2 (NuGEN). 5µg cDNA were fragmented and hybridized with Affymetrix Mouse Genome 430.2 array chips. Experiments were done using 3 independent sample sets.
Project description:The bidirectional communication between bovine oocytes and CCs is vital for functioning and development of both cell types. We used microarray to identify genes which are differentially expressed between germinal vesicle (GV)- and metaphase II (MII)-stage oocytes and CCs and those differentially expressed when oocytes mature with or without the other. We also identified genes differentially expressed between CCs at GV and MII stages. Overall design: Slaughterhouse ovaries were collected and GV-stage cumulus oocyte complexes (COCs) were aspirated. Different stages and types of oocytes and CCs were used for total RNA isolation and hybridisation on Affymetrix microarray.
Project description:Developmental competences of oocytes derived from prepubertal heifers are lower than those derived from adult counterparts. The objective of this study was to identify a range of genes associated with reduced oocyte competence that are differentially expressed between adult versus prepubertal donors. Microarray experiments were conducted using total RNA isolated from GV and MII stages oocytes collected from adult and prepubertal animals using Affymetrix GeneChip Bovine Genome Array containing 24,072 probe sets representing over 23,000 transcripts. A total of 549 and 333 genes were differentially expressed between prepubertal versus adult bovine MII and GV stages oocytes respectively. Out of these, 312 and 176 genes were up-regulated, while 237 and 157 were down-regulated in prepubertal when compared with adult MII and GV oocytes respectively. Ontological classification of the differentially expressed genes revealed that up-regulated genes in adult oocytes were involved in signal transduction, regulation of transcription DNA-dependent, and transport. Results from the present study indicated that significant number of genes were differentially expressed (>2-fold, p<0.01) between the two groups. Thus the decreased developmental competence of oocytes from prepubertal heifers may be induced due to difference in gene expression abundance as observed in our study. In conclusion, transcript abundance analyses of oocytes using microarray approach have been carried out in bovine and several other species. However, to our knowledge, this is the first study carried out to examine genes expression differential abundance in oocytes derived from perpubertal versus adult Japanese Black Cattle. Bovine 4b PP biological rep1, Bovine 78b PP biological rep2, Bovine 79 PP biological rep3 represents GV stage oocytes derived from Prepubertal (PP) heifer group, while Bovine 74b A biological rep1, Bovine 80b A biological rep2, Bovine 81 A biological rep3 represents GV stage oocytes derived from Adult (A) cow group. Bovine 7 PP biological rep1, Bovine 53 PP biological rep2, Bovine 57 PP biological rep3 represents MII stage oocytes derived from Prepubertal heifer group, while Bovine 59 A biological rep1, Bovine 70 A biological rep2, Bovine 71 A biological rep3 represents MII stage oocytes from Adult cow group. The ovaries of adult cows (Japanese black cattle) were collected from local abattoir while ovaries of prepubertal Japanese black heifers (9 to 12 months old) were collected by spay device at several commercial farms. The collected ovaries of both the adult cows and prepubertal heifers groups were transported to the research laboratory in 0.67% (w/v) NaCl solution containing 100 mg/L kanamycin sulfate (Meiji Seika, Tokyo, Japan). For both groups, cumulus oocyte complexes (COCs) from ovarian follicles 2 to 8 mm in diameter were aspirated by using an 18 gauge needle (Terumo co, Tokyo, Japan) attached to a 5 ml disposable syringe (Nipro, Osaka, Japan). After collection, the COCs were washed twice with Tyrode-lactate-pyruvate-polyvinylalcohol (Hepes-TLP-PVA) and TCM 199 (Invitrogen, Gibco, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (maturation medium). Only COCs with evenly granulated cytoplasm surrounded by multiple layers of compact cumulus cells were used in all the experiments. The COCs (70 to 80) were placed in 200 µL drop of the maturation medium in petri dish (35x10mm, Becton Dickinson Labware, Oxnard, CA, USA) covered with paraffin liquid (Nacalai Tesque Inc, Kyoto, Japan) and cultured at 38.5°C in a humidified atmosphere of 5% CO2 in air for 20 to 22 h for maturation.