Fishing for genes that affect efficiency of gynogenesis in rainbow trout (Oncorhynchus mykiss)
Gynogenetic development in fish is induced by activation of eggs with irradiated spermatozoa followed by exposure of the activated eggs to the temperature or high hydrostatic pressure (HHP) shock that prevents 1st cell cleavage. Produced specimens are fully homozygous fish also known as Doubled Haploids. Gynogenetic DH individuals might be used aquaculture and developmental biology unfortunately; the potential application of DHs is limited by a rather low survival rate of such specimens. However, observed variation in the survival rates of the gynogenetic embryos originated from different clutches suggests that eggs from some females have increased ability for gynogenetic development than others. Taking into account that first 10 cell cleavages in the fish embryos rely on the maternal RNA, it is tempting to assume that the ova showing such a vast difference in potential for gynogenesis may have also had different biological characteristics including alterations in maternal gene expression profiles. If so, then genes that up- or down –regulated expression in eggs increases competence for gynogenetic development in trout might be considered as candidate genes for gynogenesis in rainbow trout. Thus, the main goal of the project is identification of genes that increase ability of rainbow trout eggs for gynogenetic development. Within the project, we tried to verify following hypotheses: 1. Eggs from different females have different potential for gynogenesis in rainbow trout. 2. Eggs with different ability for gynogenetic development with all maternal inheritance have different biological characteristics including morphology and anti-ROS enzyme activities. 3. Eggs with increased competence for gynogenesis have altered transcriptomic profiles. 4. There are some particular genes that altered expression in trout eggs enable development of gynogenetic embryos. Gynogenetic rainbow trout specimens were produced in the course of activation of eggs with UV-irradiated spermatozoa and High Hydrostatic Pressure shock (HHP) applied around 1st cell cleavage. Eggs from several females were used in the experiment. Survival rates of gynogenetic rainbow trout was monitored since fertilization. Quality of eggs was examined by assessment of their morphology and activity of anti-ROS (reactive oxygene species) enzymes. Transcriptome of eggs showing increased and decreased developmental competence for gynogenesis was analyzed using RNA-seq approach and results compared to find out any alterations related to survival of gynogenetic trout. Overall design: Rainbow trout eggs originated from 7 females were stripped. Eggs from each female were divided into two batches and fertlized separately by the UV-irradiated (G) and non-irradiated sperm to develop into gynogenetic (G) and normal (F) (control) offspring. Samples of eggs from each female and each batch were collected and stored in RNA later for the RNA extraction. Eggs fertilized with UV-irradiated sperm were exposed for high hydrostatic pressure shock to recover diploid state in gynogenetically activated eggs. Provided within such procedure zygotes were fully homozygoous gynogenetic Doubled Haploids with only maternal chromosomes. Eggs from both, gynogenetic and control groups were incubated to monitor development of embryos. Survival before hatching and after hatching was assessed. RNA was extracted from collected eggs and their transcriptomic profiles have been provided. Expression profiles of maternal RNA in eggs from each batch and group were compared with the survival of embryos to find out any connection between increased efficiency of gynogenesis and expression of particular maternal transcripts. Genes with altered expression in relation to high survival of Doubled Haploids would be treated as candidate genes realted with increased competence of eggs for gynogenesis. <<<<<<<<<< Grant number: 2020/39/B/NZ9/00865 Title: Fishing for genes that affect efficiency of gynogenesis in rainbow trout (Oncorhynchus mykiss) Name of the funding source: National Science Center (Poland) >>>>>>>>>>