Insights into Early Ontogenesis of Salmo salar: RNA Extraction, Housekeeping Gene Validation and Transcriptional Expression of Important Primordial Germ Cell and Sex-Determination Genes

SIMPLE SUMMARY: The development of primordial germ cells (PGCs) and sex determination (SD) is governed by a complex interplay of genes that can be targeted to hinder sexual development in fish. Obtaining high-quality ribonucleic acid (RNA) from Atlantic salmon embryos, especially after fertilization, can be extremely challenging due to the presence of a substantial amount of yolk. The objective of this research was to extract high-quality RNA from developing salmon embryos for use in downstream applications. The study also aimed to validate different housekeeping genes (HKGs) for a quantitative polymerase chain reaction (qPCR)-based assessment of PGC and SD genes during developmental stages in salmon. We isolated RNA from the developing embryos, and we present the validation of HKGs as well as the mRNA expression levels of important PGC and SD genes during the fertilization to hatching stage. The findings of this study could prove beneficial for researchers seeking to extract high-quality RNA from salmonids. Moreover, the transcript results may offer insights not only into the function of transcripts at specific developmental stages but also in identifying the stage with the highest expression levels, during which treatment to disrupt the function of the transcripts and ultimately the growth of PGCs could be administered. ABSTRACT: The challenge in extracting high-quality RNA impedes the investigation of the transcriptome of developing salmonid embryos. Furthermore, the mRNA expression pattern of important PGC and SD genes during the initial embryonic development of Salmo salar is yet to be studied. So, in the present study, we aimed to isolate high-quality RNA from eggs and developing embryos to check vasa, dnd1, nanos3a, sdf1, gsdf, amh, cyp19a, dmrt1 and foxl2 expression by qPCR. Additionally, four HKGs (GAPDH, UB2L3, eEf1a and β-actin) were validated to select the best internal control for qPCR. High-quality RNA was extracted, which was confirmed by spectrophotometer, agarose gel electrophoresis and Agilent TapeStation analysis. UB2L3 was chosen as a reference gene because it exhibited lower intra- and inter-sample variation. vasa transcripts were expressed in all the developmental stages, while dnd1 was expressed only up to 40 d°C. Nanos3a was expressed in later stages and remained at its peak for a shorter period, while sdf1 showed an irregular pattern of mRNA expression. The mRNA expression levels of SD genes were observed to be upregulated during the later stages of development, prior to hatching. This study presents a straightforward methodology for isolating high-quality RNA from salmon eggs, and the resulting transcript profiles of significant PGC and SD genes in S. salar could aid in improving our comprehension of reproductive development in this commercially important species.