De novo transcriptome analysis to search for sex-differentiation genes in the Siberian sturgeon

The sturgeon family includes many species that are lucrative for commercial caviar production, some of which face critical conservation problems. The purpose of this study was to identify genes involved in gonadal sex differentiation in sturgeons, contributing to our understanding of the biological cycle of this valuable species. A high-quality de novo Siberian sturgeon gonadal transcriptome was built for this study using gonadal samples from undifferentiated fish at 3, 5, and 6 months of age; recently sex-differentiated fish at 9 months of age; and immature males and females at 14-17 months of age. Undifferentiated fish were sexed after validation of forkhead box L2 (foxl2) and cytochrome P450, family 19, subfamily A, and polypeptide 1a (cyp19a1a) as sex markers, and the transcriptomes of the 3-month-old undifferentiated fish, 5-6-month-old future females, and 5-6-month-old putative males were compared. The ovarian program was associated with strong activation of genes involved in estrogen synthesis (cyp19a1, foxl2, and estradiol 17-beta-dehydrogenase 1), stem-cell niche building and regulation, and sex-specific nerve cell development. The genes related to the stem-cell niche were: (1) the family of iroquois-class homeodomain proteins 3, 4, and 5 (irx3, irx4, irx5-1, irx5-2, and irx5-3), which are essential for somatic-germ cell interaction; (2) extracellular matrix remodeling genes, such as collagen type XXVIII alpha 1 chain and collagen type II alpha 1 chain, matrix metalloproteinases 24-1 and 24-2, and NADPH oxidase organizer 1, which, along with the somatic cells, provide architectural support for the stem-cell niche; and (3) mitogenic factors, such as lim homeobox 2, amphiregulin, G2/M phase-specific E3 ubiquitin-protein ligase, and connector enhancer of kinase suppressor of ras 2, which are up regulated in conjunction with the anti-apoptotic gene G2/M phase-specific E3 ubiquitin-protein ligase suggesting a potential involvement in regulating the number of germ cells. Genes related to sex-specific nerve cell developments were: the neurofilament medium polypeptides, the gene coding for serotonin receptor 7, 5-hydroxytryptamine receptor 7; neurotensin, isoform CRA-a, the neuron-specific transmembrane protein Delta/Notch-like epidermal growth factor-related receptor; and insulinoma-associated protein 1. The putative testicular program was poorly characterized by elements of the immune response. The classic markers of maleness were not specifically activated, indicating that testicular differentiation occurs at a later stage. In sum, the ovarian program, but not the testicular program, is in place by 5-6 months of age in the Siberian sturgeon. The female program is characterized by estrogen-related genes with well-established roles in gonadal differentiation, but also by several genes with no previously-described function in the ovarian development of fish. These newly-reported genes are involved in stem-cell niche building and regulation as well as sex-specific nerve development.