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

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. Overall design: Released oocytes were obtained in the context of a study carried out by de Sousa and co-workers (de Sousa et al., 2014, submitted) focusing on expression profiles characterizing oocytes with different quality level. Spawning of females was induced by thermal stimulation, consisting of exposure to alternate cycles of 29°C (1 hour) and 5°C (30 minutes). As each female began to spawn, it was removed from the spawning tank and transferred to an individual spawning beaker with filtered seawater at the same temperature. Once spawning was completed, the obtained oocytes were gently washed into a clean glass. The remaining oocytes of each female spawning were mixed with a sperm suspension (from 7 males) by gentle agitation, aiming to obtain around 10 spermatozoids by oocyte in a microscopic view. Moreover, to evaluate the quality of the collected oocytes, the D-larval rate (ratio between the number of free swimming larvae at 48h post fertilization and the number of starting eggs) of each eggs batch was registered. Based on that observation, oocytes were divided into two experimental groups: spawned oocytes with low hatching rate (LHR, 5%-21% of D-larval rate) and spawned oocytes with medium hatching rate (MHR, 40%-47% of D-larval rate). In addition, gonads from five females were dissected and oocytes were collected through a practice known as gamete stripping. As the name indicates, this procedure involves removal of gametes from gonad tissue. Briefly, fully ripe gonads were slashed repeatedly with a scalpel and washed with filtered seawater to harvest the gametes. Finally, sex determination and oocytes appearance were achieved through microscopy examination. About 20,000 oocytes for each spawning/stripping were collected and filtered in a 40 µm sieve. The oocytes were transferred into an Eppendorf tube and, after a short spin, the seawater was removed. To remove salts, the pellet of oocytes was re-suspended with a solution of ammonium formate 3%, which was immediately removed after a short spin. Then the oocytes were included in 1.5 ml of Extract-all solution (Eurobio) and preserved in liquid nitrogen until RNA isolation. RNA quality and integrity were 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. Feature Extraction 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. The fluorescence values were normalized by performing a quantile normalization in R statistical software. Statistical analyses were performed on 31,862 out of 59,951 probes with signal higher than background in at least 5 out of 15 target samples. A log base 2 transformation was applied to all expression values and finally, the parametric Combat algorithm was implemented in R in order to adjust for the known between-experiments batch effect.