Characterization of the European Sea Bass (Dicentrarchus labrax) Gonadal Transcriptome During Sexual Development

The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17β10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species

Improved biomarker discovery through a plot twist in transcriptomic data analysis

26 pages, 9 figures, 3 tables, supplementary information https://doi.org/10.1186/s12915-022-01398-w.-- Availability of data and materials: All data generated or analyzed during this study are included in this published article, its supplementary information files and publicly available repositories. The datasets analyzed are available at the GEO database with the accession numbers GSE115841 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE115841), GSE117590 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi), and GSE116278 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE116278)Background: Transcriptomic analysis is crucial for understanding the functional elements of the genome, with the classic method consisting of screening transcriptomics datasets for differentially expressed genes (DEGs). Additionally, since 2005, weighted gene co-expression network analysis (WGCNA) has emerged as a powerful method to explore relationships between genes. However, an approach combining both methods, i.e., filtering the transcriptome dataset by DEGs or other criteria, followed by WGCNA (DEGs + WGCNA), has become common. This is of concern because such approach can affect the resulting underlying architecture of the network under analysis and lead to wrong conclusions. Here, we explore a plot twist to transcriptome data analysis: applying WGCNA to exploit entire datasets without affecting the topology of the network, followed with the strength and relative simplicity of DEG analysis (WGCNA + DEGs). We tested WGCNA + DEGs against DEGs + WGCNA to publicly available transcriptomics data in one of the most transcriptomically complex tissues and delicate processes: vertebrate gonads undergoing sex differentiation. We further validate the general applicability of our approach through analysis of datasets from three distinct model systems: European sea bass, mouse, and human. Results: In all cases, WGCNA + DEGs clearly outperformed DEGs + WGCNA. First, the network model fit and node connectivity measures and other network statistics improved. The gene lists filtered by each method were different, the number of modules associated with the trait of interest and key genes retained increased, and GO terms of biological processes provided a more nuanced representation of the biological question under consideration. Lastly, WGCNA + DEGs facilitated biomarker discovery. Conclusions: We propose that building a co-expression network from an entire dataset, and only thereafter filtering by DEGs, should be the method to use in transcriptomic studies, regardless of biological system, species, or question being considered.NS was supported by a Spanish Ministry of Science (SMS) predoctoral scholarship (BES-2017-079744). Research was supported SMS grant no. PID2019-108888RB-I00 to FP. Also with funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

La lubina europea muestra el camino para comprender los mecanismos de determinación del sexo en peces

Por primera vez los científicos demuestran como predecir el sexo de un animal sin cromosomas sexuales ni gen dominante determinante del sexoPeer reviewe

Weighted correlation network analysis and transcriptome dynamics during early gonadal differentiation of the European sea bass (Dicentrarchus labrax)

Next Generation Sequencing (NGS 2018), 9-11 April 2018, Barcelona, SpainPeer Reviewe

Nuevos estudios sobre la expresión génica de la lubina

En un estudio acabado de publicar en Marine Biotechnology se presenta un estudio de los cambios transcriptómicos en las gónadas de la lubina durante el proceso de diferenciación sexualPeer reviewe

Gonadal transcriptomic dynamics during sex differentiation across vertebrates

XIII Congress of the Iberian Association of Comparative Endocrinology - XIII Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), 16-17 September 2021Gonadal sex differentiation is the process by which the bi-potential gonads transform into testis or ovaries after sex determination. A large number of genes and pathways involved in this process have been discovered and are fairly conserved across vertebrates. However, the temporal and relative expression of these genes may be different in the major five vertebrate groups, although a comparative study has never been carried out. To investigate these possible differences, we used both published and unpublished studies concerning sex differentiation in different vertebrate species including the European sea bass, platyfish, red-eared slider turtle, frog, chicken, and mouse. First, we determined the differentially expressed genes, within each sex, between the beginning and towards the end of sex differentiation. Male- and female-enriched genes were defined based on their higher expression in males and females, respectively, towards the end of sex differentiation. Next, we compared the transcriptome dynamics and specific gene expression profiles of a set of key genes we selected based on their implication on gonadal sex differentiation. The major insights of this study are: 1) We found that the percentage of male- and female-enriched genes that were either upregulated or downregulated during ovarian differentiation and testicular differentiation greatly varied across the different vertebrate species analyzed, including the two fish species; 2) Sex differentiation can be achieved with different degrees of upregulation of pro-male or pro-female genes, depending on the sex, with or without concomitant downregulation of genes of the opposite sex; 3) In some cases, rather than different patterns across sexes, differences were observed in the degree of upregulation vs downregulation of the pro-male and pro-female genes. Taken together, these results show that there are important differences in the temporal expression dynamics between representative species of the major groups and that sex differentiation is achieved by different regulatory mechanismsResearch funded by the Ministry of Science and Innovation ‘Epipure’ (PID2019-108888RB-100) grant to F

Descripció del transcriptoma gonadal del llobarro (Dicentrarchus labrax) al llarg de differents estadis del desenvolupament

XV Jornada de Biologia de la Reproducció, 21 de juny de 2017, BarcelonaPeer Reviewe

Development of epigenetic biomarkers to aid in animal production, natural resource management and the assessment of the effects of climate change

XI Annual Chromatin and Epigenetics symposium : XI Jornada de Cromatina i Epigenètica, organizada por la Secció de Cromatina i Epigenètica de la Societat Catalana de Biologia (SCB) y el Barcelona Chromatin Club (BCC)In recent years, the progressive lowering of sequencing and analytical costs has prompted the development of epigenetic biomarkers for the early diagnosis and monitoring of several diseases, particularly cancer. In a non-biomedical context, epigenetic biomarkers are gaining interest in the areas of animal production, natural resource management and in the assessment of the effects of climate change. This reflects the increasing interest in the development of epigenetic biomarkers for ecology- and evolution-oriented studies. Among epigenetic biomarkers, those based on DNA methylation changes are favored because they can be easily measured, respond quickly to environmental influences and, as accumulating evidence shows, can persist across generations. Here, we will present our studies aimed at: 1) understanding the contribution of epigenetic changes (epimutations) to the early stages of domestication of farm animals, 2) the development of the first piscine epigenetic clock for age determination in fish, 3) the identification of metastable epialleles as footprints of the effects of climate change in marine life, and 4) the development of epigenetic biomarkers to aid in broodstock selection in aquacultur

Descripció del transcriptoma gonadal del llobarro (Dicentrarchus labrax) al llarg de diferents estadis del desenvolupament

XV Jornada de Biologia de la Reproducció, 21 de juny de 2017.-- 5 pages, 4 figures[EN] The European sea bass (Dicentrarchus labrax) is an economically important farmed species around the Mediterranean for which the demand is rapidly increasing. Nevertheless, under captivity male-biased populations still occur. In this species, since the sex ratio is determined by several loci and influenced by environmental factors such as temperature. Such bias in favour of males negatively affects production because females outgrow males by ~30% in weight. In order to better understand the sex determination and differentiation processes of the European sea bass we conducted a comprehensive study on the gonadal transcriptome at three different stages of development: at 110, 250 and 350 days post fertilization (dpf), using a custom-made microarray for the target species. Microarray results were validated by real time PCR (qPCR). A total of ~44.000 probes were obtained. The statistical Significance Analysis of Microarrays (SAM) test determined 113 common differentially expressed (DE) genes when comparing ovaries with testis along the three stages of gonadal development, 98 genes DE between 110 and 250 dpf, and 1,496 DE genes between 250 and 350 dpf. The expression profile of 16 canonical genes important for reproduction and sex differentiation in fish was studied as well as pathways known to be related with male or female gonad differentiation. The proportion of expressed transcription factors (TFs) was also compared between gonads along development. Furthermore, a set of genes were identified to be DE between ovaries and testis that significantly changed throughout development, providing a list of candidate genes enrichment for further investigation[CAT] El llobarro (Dicentrarchus labrax) és una espècie de peix produïda en països de la conca Mediterrània, econòmicament important i del qual la demanda està augmentant ràpidament. No obstant, sota condicions de captivitat, encara s'obtenen poblacions amb major proporció de mascles. En aquesta espècie la proporció de sexes depèn d'uns quants loci i és influïble per factors mediambientals com la temperatura. Aquestes proporcions actuen en detriment de la producció, ja que les femelles creixen un ~30% més que els mascles. Per tal de comprendre millor els processos de determinació i diferenciació del sexe del llobarro, es va realitzar un estudi exhaustiu del transcriptoma gonadal a tres estadis diferents del desenvolupament: a 110, 250 i 350 dies després de la fecundació (dpf) mitjançant un microarray dissenyat específicament per aquesta espècie. Els resultats foren validats per PCR a temps real (qPCR). Es van obtenir un total de ~44.000 sondes. L'anàlisi de Significació Estadística de Microarrays (SAM), determinà 113 gens comuns diferencialment expressats (DE) quan es comparaven ovaris contra testicles al llarg de les tres etapes de desenvolupament, 98 gens DE comuns a 110 i 250 dpf, i 1.496 a 250 i 350 dpf. A més, es van identificar un conjunt de gens DE entre els ovaris i els testicles i que canvien significativament al llarg del desenvolupament gonadal. El perfil d'expressió de 16 gens canònics importants per a la reproducció i la diferenciació sexual en peixos va ser estudiada, així com el de les vies metabòliques relacionades. La proporció de factors de transcripció expressats (TFs) també es va comparar entre les gònades masculines i femenines. Aquesta llista proporciona informació de gens candidats per futures investigacionsAquest treball ha estat finançat pel projecte “Aquagenomics”, Consolider-Ingenio 2010 (ref. CDS2007-0002) concedit a FPPeer Reviewe

Increaased biomarker discovery through a twist on the analysis of trasncriptomic data during sexual development in the European sea bass, mouse and humans

30th CECE & 9th ISFE : Joint Conference of the European Society for Comparative Endocrinology and of the International Society for Fish Endocrinology, 4-8 September 2022, Faro, PortugalThe transcriptomic analysis is crucial to understanding the functional elements of the genome. The classic method is screening for differentially expressed genes (DEGs). Since 2005 weighted gene co-expression network analysis (WGCNA) has emerged as a powerful method to explore the relationships between genes. However, a method combining the two, i.e., filtering the transcriptome dataset by DEGs or other criteria and then applying WGCNA (DEGs+WGCNA), has become common. This is of concern because it can the resulting underlying architecture of the network and lead to wrong conclusions. Here, we explore a plot twist to transcriptome data analysis: combining WGCNA, exploiting the whole dataset and not affecting the topology of the network, with the strength and relative simplicity of DEGs analysis (WGCNA+DEGs). We tested WGCNA+DEGs against DEGs+WGCNA to publicly available transcriptomic data in one of the most transcriptomically complex tissues and delicate process: vertebrate gonads undergoing sex differentiation. We generalized our results in three systems: sea bass, mouse, and human. Despite the diversity of sex-determining systems in vertebrates, a common feature is that a complex network of multiple regulatory interactions of genes associated with male and female pathways are expressed simultaneously as opposing forces. Although the gene networks involved in converting the gonads into ovaries or testis are relatively conserved during sex differentiation, the temporal and relative expression, as well as the relative position within the network, are different across vertebrates. The use of network properties and tools could contribute to better resolve such complex networks. As a result, WGCNA+DEGs clearly outperformed DEGs+WGCNA. First, the networks model fit, node connectivity measures as well as other network statistics improved, the number of modules and key genes retained increased and, importantly, the GO terms of biological processes provided a more meaningful representation of the biological question under consideration. Lastly, WGCNA+DEGs facilitated the discovery of a relevant biomarker. We propose that building a co-expression network from the entire dataset, and only thereafter filtering by DEGs, should be the method to use in transcriptomic studies, regardless of biological system, species or question being consideredPeer reviewe