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SInsights into molecular features of Venerupis decussata oocytes: a microarray-based study.

ABSTRACT: The European clam, Ruditapes decussatus (Linnaeus, 1758), is a bivalve mollusc of the family Veneridae native to the European Atlantic and Mediterranean coastal waters. Its production is exclusively based on natural recruitment, which is subject to high annual fluctuations due to adversely affected by pollution and other environmental factors. In the present study, oocytes from 15 females were collected in Ria de Aveiro (Western coast of Portugal) and microarray analysis was performed in order to investigate gene expression profiles characterizing naturally released oocytes and ovarian oocytes obtained by stripping. Released oocytes were obtained in the context of a study carried out by de Sousa and co-workers (GSE54954: de Sousa et al., 2014, submitted) focusing on expression profiles characterizing oocytes with different quality level. In particular, to compare stripped and spawned oocytes, a total of 10 samples (5 with bad/intermediate hatching rate and 5 with good hatching rate) out of the 25 analyzed in the previous study (GSE54954), and 5 new pools of stripped oocytes, have been analyzed by microarray analyses.

ORGANISM(S): Ruditapes decussatus

PROVIDER: GSE58906 | GEO | 2014/11/05

SECONDARY ACCESSION(S): PRJNA253844

REPOSITORIES: GEO

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Project description:The European clam, Ruditapes decussatus (Linnaeus, 1758) is a bivalve mollusc of the family Veneridae native to the European Atlantic and Mediterranean coastal waters. Its production is exclusively based on natural recruitment, which is subject to high annual fluctuations due to adversely affected by pollution and other environmental factors. Microarray analyses have been performed in four gonadal maturation stages of two higly productive Portuguese wild populations (Ria Formosa in South and Ria de Aveiro in North) characterized by different responses to spawning induction. Twenty clams (Ruditapes decussatus)were monthly sampled between July 2011 and July 2012 in two different Portuguese wild populations: Ria Formosa and Ria de Aveiro. At each sampling date the gonads of the collected clams were immediately dissected for RNA extraction and fixed for histological analysis. Total RNA was isolated using Extract-all (Eurobio) procedure. RNA quality and integrity was controlled on the Agilent bioanalyzer using RNA nanochips and Agilent RNA 6000 nanoreagents (Agilent Technologies, Waldbronn, Germany). RNA concentrations were measured at 260 nm using a ND-1000 spectrophotometer (Nanodrop Technologies) using the conversion factor 1 OD = 40 mg/mL total RNA. Samples were stored at -80°C until further use. Gene expression profiling was performed using an R.decussatus oligo-DNA microarray of 59,951 probes based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA at a resolution of 2 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. FeatureExtraction v10.7.3.1 used XDR ID to link the pairs of scans together automatically when extracting data. The signal left after all the FE processing steps have been completed is ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.

Project description:Since the identification of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) as the main maturation-inducing hormone (MIH) in Japanese eel A. japonica, female European eels have been injected with this steroid to induce oocyte maturation and ovulation. However, the females injected with DHP need to ovulate rapidly following the DHP injection to release good quality eggs. In this study, we compared treatment of progesterone (P) with DHP treatment as control, in vitro, to determine dose-response effects, and in vivo, at a single dose. Females were first matured by the routine protocol of weekly injections with carp pituitary extract (CPE at 20 mg kg-1) until a significant oocyte hydration response occurred as indicated by an increase of the body weight and oocytes displaying germinal vesicle migration. For the in vitro experiment, ovarian tissue was extracted and placed in culture plates containing hormone-free medium and media supplemented with the treatment (P: 10, 100, 1,000 ng.mL-1; DHP: 1, 10 and 100 ng.mL-1). Before and after incubation for 12 and 18 h, the folliculated oocytes were sampled for microscopy and gene expression analysis. For the in vivo experiment, females were either injected with P or DHP at a dose of 2 mg.kg-1 to assess their effects on ovulation and reproductive success. At the moment of stripping, eggs were sampled for RNA-sequencing to compare P vs. DHP stripped eggs with focus on genes marking gamete quality. Remaining eggs were fertilized and larval viability was recorded. Both P and DHP were able to induce germinal vesicle breakdown in vitro (P at 100 and 1,000 ng mL-1 ;DHP at 10 ng and 100 ng mL-1). RNAseq results reflected similar P and DHP effects on the expression of genes involved in egg quality aspects. Also expression of genes involved in oocyte maturation and ovulation was similar for both P and DHP treatment. Females injected with either P or DHP ovulated, released eggs and were equally able to produce larvae without any differences in reproductive success. Our results support the conclusion that P and DHP have similar competence as MIH which may be because P binds to the membrane progestin receptors with similar capacity as DHP does. P is more attractive to apply as the price is 3,000 times lower than the price of DHP.

Project description:Background: Non-genetic disease inheritance and offspring phenotype is substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2 dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. Results: In this study we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. Conclusions: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes, and that this activity is conserved in the mouse and human germlines.

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SInsights into molecular features of Venerupis decussata oocytes: a microarray-based study.

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