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Antisense RNA technology induces the loss of function of steroidogenic factor 1 leading to abortion of tilapia

ABSTRACT: As a precise and efficient genetic engineering method, gene editing technology for animals and plants will gradually move towards large-scale applications, and will have a disruptive impact on the breeding of related traits. By using antisense RNA interference technology to interfere with the mRNA expression and protein translation of the SF-1 gene, we have successfully cultivated tilapia with defective gonadal development. The results of the study showed that the weights of male and female fish in the experimental group were significantly higher than those of the control group. The gonadal weight and gonadal index of the experimental group were significantly lower than those of the control group and the negative control group. HE staining showed that the number of spermatogonia in the testis tissue of the experimental group was significantly reduced, the cavity in the seminiferous tubules was significantly enlarged and increased, the number of sperm cells was significantly decreased, and the number of sperm was relatively rare. However, a large number of stage V oocytes accumulated in the ovarian tissue of female fish in the experimental group, and many of them were gray-white. The mRNA and protein expression levels of SF-1 gene in the gonadal tissues were extremely significantly lower than those of the control and negative controls. The combined analysis of transcriptome and proteome showed that Steroid hormone biosynthesis, Arachidonic acid metabolism and Cell adhesion molecules are involved in the regulation of the gonads of male fish after SF-1 gene silencing. However, ECM-receptor interaction, Retinol metabolism, Nicotinate and nicotinamide metabolism and cytochrome P450 may be involved in the regulation of female fish SF-1 gene silencing. SF-1 silencing may cause developmental defects in the external reproductive organs of male and female fishes and inhibit the development of gonadal tissues through different regulatory pathways. This provides data support for the further functional analysis of SF-1 on fish and the treatment of abnormal human SF-1 expression.

ORGANISM(S): Oreochromis niloticus

PROVIDER: GSE161135 | GEO | 2023/11/01

REPOSITORIES: GEO

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Project description:As a precise and efficient genetic engineering method, gene editing technology for animals and plants will gradually move towards large-scale applications, and will have a disruptive impact on the breeding of related traits. By using antisense RNA interference technology to interfere with the mRNA expression and protein translation of the SF-1 gene, we have successfully cultivated tilapia with defective gonadal development. The results of the study showed that the weights of male and female fish in the experimental group were significantly higher than those of the control group. The gonadal weight and gonadal index of the experimental group were significantly lower than those of the control group and the negative control group. HE staining showed that the number of spermatogonia in the testis tissue of the experimental group was significantly reduced, the cavity in the seminiferous tubules was significantly enlarged and increased, the number of sperm cells was significantly decreased, and the number of sperm was relatively rare. However, a large number of stage V oocytes accumulated in the ovarian tissue of female fish in the experimental group, and many of them were gray-white. The mRNA and protein expression levels of SF-1 gene in the gonadal tissues were extremely significantly lower than those of the control and negative controls. The combined analysis of transcriptome and proteome showed that Steroid hormone biosynthesis, Arachidonic acid metabolism and Cell adhesion molecules are involved in the regulation of the gonads of male fish after SF-1 gene silencing. However, ECM-receptor interaction, Retinol metabolism, Nicotinate and nicotinamide metabolism and cytochrome P450 may be involved in the regulation of female fish SF-1 gene silencing. SF-1 silencing may cause developmental defects in the external reproductive organs of male and female fishes and inhibit the development of gonadal tissues through different regulatory pathways. This provides data support for the further functional analysis of SF-1 on fish and the treatment of abnormal human SF-1 expression.

Project description:The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female -induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.

Project description:SF-1, a transcription factor belonging to the nuclear receptor superfamily, has a pivotal role for adrenogonadal development in humans and mice. A constant feature of childhood adrenocortical tumors (ACT) is SF-1 amplification and overexpression. Using an inducible cellular system, here we show that SF-1 overexpression increases human adrenocortical cell proliferation through opposing effects on cell cycle and apoptosis. SF-1 overexpression also selectively modulates steroidogenesis, reducing cortisol and aldosterone secretion. We identified a novel pro-apoptotic factor for adrenocortical cells, NOV/CCN3, whose levels are significantly reduced by SF-1 overexpression in human adrenocortical cells and are also reduced in primary adrenal tumors. Moreover, Sf-1 overexpression triggers adrenocortical hyperplasia and tumor formation in mice. These tumors express gonadal markers and activated Stat3. Our studies reveal the critical role of SF-1 gene dosage for adrenocortical tumorigenesis and constitute a rationale for the development of drugs targeting SF-1 transcriptional activity for ACT therapy. Keywords: comparative gene expression Gene expression profiles were analyzed by two-color microarrays in transgenic mice from line #56 heterozygous or homozygous for the transgene, male or female and of different ages. Profiles from each transgenic mouse were directly compared to a sex- and age-matched wild-type littermate. For each experimental point, two technical replicates (dye-swaps) were examined. Array # Age Sex Transgene copy # Cy3 Cy5 16682 10 days M 1 WT transgenic 16479 10 days M 1 transgenic WT 16683 10 days M 1 WT transgenic 16625 10 days M 1 transgenic WT 16685 10 days F 1 WT transgenic 16680 10 days F 1 transgenic WT 16516 4 months M 2 WT transgenic 16452 4 months M 2 transgenic WT 16517 4 months M 2 WT transgenic 16453 4 months M 2 transgenic WT 16518 4 months F 2 WT transgenic 16475 4 months F 2 transgenic WT

Project description:Gonad is a bipotential tissue that depending on the genetic and environmental inputs its final phenotype will be determined. Gonadal development is a very complex process because from an undifferentiated gonad the future testis or ovary will be formed. To better understand the complexity of the gonad plasticity throughout development we explored the transcriptomic landscape of gonads from undifferentiated to juvenile fish. For this study we have used turbot (Scophthalmus maximus) that presents one of the most remarkable sexual dimorphism in cultured fish species. With the aim of identifying gene patterns for each gonad developmental stages a microarray species-specific enriched by reproduction- and immune-related genes was assessed. Further genes with sex-differential mRNA expression were mapped in the sex- and growth-QTL (quantitative trait loci) markers. Data revealed that gonadal transcriptome prevail in front of fish biometry as it was possible to classify gonads according to sexual development status by using gene expression profiles rather than their age or size. The same classification was observed with a selection of a subset of reproduction-related genes showing the importance of gene profiles for each of the developing gonadal stage. Overall microarray data showed the importance of the level of expression (intensity) rather than the number of genes found in each development period. Larger numbers of differentially expressed genes (DEG) were found in the differentiating gonads when compared to ovaries and much smaller number resulted when compared to testis. However, more number of DEG was found in testis when compared to ovaries. The earliest presence of cyp19a1a, the key female sex differentiation gene, was found at 90 dpf together with its transcription factor foxl2 showing at the same time a positive correlation with other genes related to extracellular matrix (ecm2), immune system (irf5) or methylation (sall1). The presence of male-related genes like sox9 was evidenced at early developmental stages (70 and 90 dpf) while expression of dmrt3 started not earlier than 150 dpf, coinciding when larger number of male-related genes were found. All developmental stages presented genes related to immune system and epigenetic mechanisms revealing their importance during gonadal development. Sex-QTLs showed larger amount of sex-differential transcripts in the linkage group 6 and 21 both in males and females but transcripts mapped near growth-QTLs were only found in females. Here it is presented the first assessment of the expression profiles observed in the gonads at different developmental stages by identifying genes responsible for male or female traits. It has been identified gene clusters for each developmental stages and gene network interactions for each sexual phenotype. The expression of known male and female-related genes were precisely described throughout gonadal development as well as the expression of other genes with an unknown reproduction function which have been identified for the first time during sexual development process. In addition, it was possible to localize some of these genes on the sex- and growth-QTLs. The data presented here can help to better understand the reproduction system in cultured fish species and improved selection breeding programs to increase their productivity.

Project description:Background: In the life history of Daphnia, the reproductive mode of parthenogenesis and sexual reproduction alternate in aquatic ecosystem, which are often affected by environmental and genetic factor. Although several functional genes on reproductive transition of Daphnia had been determined, molecular mechanism on the reproductive mode of Daphnia are still not known well, including differentially expressed genes in different developmental stages. Results: In this study, four developmental stages, juvenile female (JF), parthenogenetic female (PF), sexual female (SF) and male (M), of D. similoides sinensis were performed for transcriptome sequence, and candidate genes related to male sex determination were screened. A total of 110437 transcripts were obtained and assembled into 22996 unigenes. In the four developmental stages (JF, PF, SF and M), the number of unique unigenes is respectively 2863, 445, 437 and 586, and the number of common unigenes is 9708. The differentially expressed genes (DEGs) between male and other three female stages (M vs JF, M vs PF and M vs SF) were obtained. The GO gene enrichment analysis showed that the up-regulated genes in male were mainly enriched in hydrolase activity and peptidase activity. Thirty-six candidate genes related to male sex determination in male were significantly higher expression than those in the other three stages, including one Doublesex (Dsx) gene, one laminin gene, five trypsin genes and one serine protease genes, and one chitin synthase gene and two chitinase genes. In addition, in D. similoides sinensis male, the relative expression levels of two genes (Dsx1, antp) related to male sex determination observed in other Daphnia species were also significantly higher than those in other developmental stages. Conclusions: Our results showed that thirty-six candidate genes may involve in sex differetiation of D. similoides sinensis male, and it will provide a reference for further exploring the functional genes related to sex determination mechanism in Daphnia species. Moreover, according to previous investigations, we thought that the expression level of functional genes may be related to the development stage of organisms, and may be also affected by different Daphnia species.

Project description:BACKGROUND: The ultra-high density intensive farming model of grass carp (Ctenopharyngodon idellus) may elicit inhibite the growth, decline flesh quality and disease resistance of fish. The quality degradation and excessive fat accumulation in cultured C. idellus have long been attributed to possible alterations in the lipid metabolism of fish muscle tissues as a result of over-nutrition from artificial diets. To investigate the effects of different diets on fish muscle quality, a large-scale metabolomics study was performed on 200 tails of C. idellus. FINDINGS: The experimental fish were divided into four test groups based on sex and diets - female artificial feed (FAF), female grass feed (FGF), male artificial feed (MAF) and male grass feed (MGF). After a 4-month rearing period, the AF group showed significantly higher total mass of muscle fat (P < 0.01), with the FAF group being the highest. Metabolomics profiling based on liquid chromatography-mass spectrometry (LC-MS) revealed distinctive patterns of clustering according to the four groups. Overall, artificial feeding was associated with higher concentrations of docosapentaenoic acid (DPA), dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (ARA); whereas grass-feeding was associated with elevated n-3 unsaturated fatty acids (n-3 UFAs), such as eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA) and gamma-linolenic acid (GLA). Some sex-specific markers, such as docosahexaenoic acid (DHA) was only found in male samples, with higher levels in the MAF group. Metabolic pathway analyses using both targeted (MetaboAnalyst) and untargeted (mummichog) approaches consistently revealed that the arachidonic acid metabolism and steroid hormone biosynthesis pathways are significantly different between AF and GF groups. CONCLUSIONS: Our results suggested that grass is a better source of diet fatty acid and protein when compared to artificial feed, because it could effectively lower triglycerides in serum, reduce fat accumulation and alter lipid compositions in fish muscle by increasing the concentrations of n-3 UFAs, leading to better nutrition and health.

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Antisense RNA technology induces the loss of function of steroidogenic factor 1 leading to abortion of tilapia

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