Transcriptome analysis of rainbow trout Oncorhynchus mykiss eggs subjected to the high hydrostatic pressure shock

The microtubule polymers of the mitotic spindle are sensitive to the hydrostatic pressure which applied to the fertilized fish eggs results in failure of the second body extrusion or/and inhibition of the first cell cleavage. Thus, the high hydrostatic pressure is applied in aquaculture to induce development of triploid sterile fish and diploidization of the gynogenetic and androgenetic haploid zygotes. It has been observed that high doses of the hydrostatic pressure applied to the fish eggs are also damaging for other cellular organelles and may affect early development of the fish embryos. The rainbow trout eggs contains maternal mRNA that is crucial for the proper development of embryos before the zygotic genome activation. So, it may be assumed that high hydrostatic pressure being damaging for the cell organelles may also affect cytoplasmic maternal transcriptome. Therefore, the main goal of the present research was to evaluate changes in the maternal transcriptome in eggs exposed for the high pressure shock.

Eggs from three females were collected in the separate plastic containers. Half of each batch of eggs were then exposed for 9000 psi of high pressure shock that lasted 3 minutes, a condition usually used for poliploidization of the genetic material in the rainbow trout eggs. Batches of the untreated eggs were kept at 4^o C. RNA from both subjected to the high pressure shock and untreated eggs was extracted using modified TRIzol protocol. RNA quality was assessed and its integrity evaluated (Agilent's RIN) and compared between treated and untreated eggs. A total of 800 ng purified RNA was used as input for TruSeq RNA Sample Prep v2 kit (Illumina). Validated and normalized libraries were eventually sequenced using TruSeq SBSv3 Sequencing kit (Illumina) to obtain approx. 25 million reads per sample.

The mean RIN values for the treated and untreated eggs were similar. In total, 162.2 million sequencing reds were obtained and about 60% were successfully mapped to the reference transcriptome. 16,243 and 16,346 expressed transcripts were detected in the treated and untreated eggs, respectively. Differences in the expression level of transcripts between untreated eggs and eggs exposed for the high pressure shock were minor, however encompassed genes involved in the response to the DNA damage and repair, cell cycle control, chromatin remodeling and regulation, regulation of DNA synthesis and transcription (IGF, RIF, CENPF, SETD2, among others). Provided results indicated that the high pressure shock applied to the rainbow trout eggs to a certain extent effected maternal mRNA and may impair processes and mechanisms crucial for the proper development of the early fish embryos before activation of the zygotic genome.

This study was supported by the National Science Centre (NCN, Poland) project number 2014/15/BNZ9/00510