Project description:Here we investigate whether the presence of germinal vesicle-stage oocytes (GV- oocytes) reflects poor oocyte developmental competence (or quality). This was a prospective, non-randomised, cohort pilot-study involving 60 patients undergoing in vitro fertilization/ intracytoplasmic sperm injection for whom complete pregnancy outcome data were available. Patients in whom GV- oocytes were retrieved (GV+) at transvaginal oocyte retrieval (TVOR) were compared with those from whom no GVs were retrieved (GV-). We found that GV+ (n = 29) and GV- (n = 31) patients were similarly aged (35.4 vs. 36.4 years; p = 0.446). GV+ patients had a mean of 2.41 ± 2.03 GVs and comparable yields of MII oocytes to GV- patients (11 ± 6.88 vs. 8.26 ± 4.84; p = 0.077). Compared with GV- patients, GV+ patients had markedly lower implantation rates (11.8% vs. 30.2%; p = 0.022) as well as oocyte utilisation rates for clinical pregnancy (2.3% vs. 6.8%; p = 0.018) and live-birth (1.9% vs. 5.7%; p = 0.029). DNA damage levels measured using ?H2AX immunostaining were not different in oocytes from women <36 years versus those ?36 years (p = 0.606). Thus, patients who have GV- stage oocytes at TVOR exhibit poor oocyte quality reflected in reduced per-oocyte pregnancy success rates and uniformly high levels of oocyte DNA damage.
Project description:We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.
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:We profiled transcriptomes from Cnot6l deadenylase knock-out mouse GV oocytes, MII eggs and 1-cell zygotes in order to analyse its function during the oocyte-to-embryo (OET) transition. Transcriptome of wild-type golden hamster GV oocytes was also profiled. Overall design: bulk RNA-seq of 20 samples: - 3 replicates of each WT and Cnot6l knock-outs in 3 mouse developmental stages (GV, MII, 1-cell) - 2 replicates of WT golden hamster GV oocytes
Project description:Background: Early embryonic development is governed by maternal transcripts stored within the oocyte during oogenesis. Transcriptional activity of the oocyte ultimately dictates its developmental potential and may be influenced by maternal age, resulting in reduced competence of oocytes derived from women of advanced age, compared with the young. In the current study, RNA-Seq was used to perform transcriptome profiling of human GV and MII oocytes derived from young and advanced maternal age women. Participants/Materials and Methods: Cumulus dissection from donated oocytes was performed. GV and MII oocytes underwent deep RNA sequencing using the SMART-Seq v4 Ultra Low Input RNA protocol (Takara-Clontech, USA) and Nextera XT DNA library preparation kit (Illumina, USA). Data processing, quality assessment and bioinformatics analysis were performed using source-software, including FastQC, HISAT2, StringTie, edgeR and DAVID. Results: Following deep single-cell RNA-Seq on GV and MII oocytes, hundreds of transcripts were significantly differentially expressed between young maternal age (YMA) and advanced maternal age (AMA) groups, with the most significant biological processes relating to mitochondrial reserves. The GV to MII transition shares common biological processes between young and AMA groups, however, some genes involved in mitochondria function were altered during ageing. A decrease in mitochondrial-related transcripts was also observed during the GV to MII transition. However, there was a much greater reduction of mitochondrial-related transcripts in MII oocytes of AMA. This observation was confirmed when YMA MII oocytes were compared with the AMA MII group with mitochondrial-related transcripts being significantly higher expressed in the YMA group, including biological processes, such as mitochondrial electron transport and ATP biosynthetic process. These results indicate a higher energy potential in YMA MII oocytes that is decreased with age. Other significantly higher biological processes in the YMA MII group include transcripts involved in the regulation of ubiquitin-dependent degradation. Lack of these transcripts could lead to a non-appropriate removal of oogenesis remnants following fertilisation in the AMA MII group. Discussion: Understanding reproductive ageing effects at the RNA level in human oocytes may reveal differences in the mechanisms regulating the GV to MII transition that impact on oocyte quality in YMA and AMA patients. Further investigations of the up-/down-regulated transcripts during ageing could guide and improved IVF outcomes for AMA patients. Overall design: Fifteen patients treated for infertility in a single IVF unit agreed to participate in this study, where 5 GV and 5 MII oocytes derived from 8, 21-26 yo women (YMA group), and 5 GV and 6 MII oocytes from 7, 41-44yo women (AMA group). These oocytes were donated by women undergoing IVF treatment for research purposes. The samples were collected within a period of 3 months. RNA was isolated and deep sequenced at the single cell level.
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: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 Overall design: 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:Maternal factors are required for oocyte maturation and embryo development. To better understand the role of DNA methyltransferase 1 (Dnmt1) in oocyte maturation and embryo development, small interfering RNA (siRNA) was conducted in porcine oocytes. In this study, our results showed that Dnmt1 localized in oocyte cytoplasm and its expression displayed no obvious change during oocyte maturation. When siRNAs targeting Dnmt1 were injected into germinal vesicle (GV) stage oocytes, Dnmt1 transcripts significantly decreased in matured oocytes (P<0.05). After Dnmt1 knockdown in GV stage oocytes, the significant reduction of glutathione content, mitochondrial DNA copy number, glucose-6-phosphate dehydrogenase activity and expression profiles of maternal factors and the severely disrupted distribution of cortical granules were observed in MII stage oocytes (P<0.05), leading to the impaired oocyte cytoplasm. Further study displayed that Dnmt1 knockdown in GV stage oocytes significantly reduced the development of early embryos generated through parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (P<0.05). In conclusion, Dnmt1 was indispensable for oocyte cytoplasmic maturation, providing a novel role for Dnmt1 in the regulation of oocyte maturation.
Project description:Successful fertilization and subsequent embryo development rely on complex molecular processes starting with the development of oocyte competence through maturation. MicroRNAs (miRNAs) are small non-coding RNA molecules that function as gene regulators in many biological systems, including the oocyte and embryo. In order to further explore the roles of miRNAs in oocyte maturation, we employed small RNA sequencing as a screening tool to identify and characterize miRNA populations present in pools of bovine germinal vesicle (GV) oocytes, metaphase II (MII) oocytes, and presumptive zygotes (PZ). Each stage contained a defined miRNA population, some of which showed stable expression while others showed progressive changes between stages that were subsequently confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR). Bta-miR-155, bta-miR-222, bta-miR-21, bta-let-7d, bta-let-7i, and bta-miR-190a were among the statistically significant differentially expressed miRNAs (p < 0.05). To determine whether changes in specific primary miRNA (pri-miRNA) transcripts were responsible for the observed miRNA changes, we evaluated pri-miR-155, -222 and let-7d expression. Pri-miR-155 and -222 were not detected in GV oocytes but pri-miR-155 was present in MII oocytes, indicating transcription during maturation. In contrast, levels of pri-let-7d decreased during maturation, suggesting that the observed increase in let-7d expression was likely due to processing of the primary transcript. This study demonstrates that both dynamic and stable populations of miRNAs are present in bovine oocytes and zygotes and extend previous studies supporting the importance of the small RNA landscape in the maturing bovine oocyte and early embryo.
Project description:To determine whether the follicle environment modulates oocyte-specific gene transcript levels in cultured oocytes and polar bodies (PBs).Animal study.Large academic research center.CD1 mice.In vitro growth of secondary mouse follicles in 0.25% or 1.5% alginate (ALG) in a three-dimensional culture system.Relative transcript levels of Gdf9, Bmp15, Nlrp5, Tcl1, and Zp3 were measured by real-time quantitative reverse transcriptase-polymerase chain reaction in oocytes during in vitro follicle development and oocyte maturation and in their first PBs after removal from metaphase II (MII) eggs.All transcripts decreased earlier in oocytes cultured in 1.5% ALG compared with 0.25% ALG. Transcript levels were lower in MII eggs cultured in 1.5% ALG compared with in 0.25% ALG. All genes were expressed in PBs, and transcript levels were lower in PBs cultured in 1.5% ALG compared with in 0.25% ALG. Abundance of all transcripts was lower in PBs than in their sibling oocytes.Local follicle environment modulates oocyte-specific gene expression in the oocyte and first PB. There is a significant difference in the transcript levels of oocyte-specific genes in PBs of 1.5% versus 0.25% ALG that correlates with ovarian environment-related decreases in oocyte competence.