Transcriptomes of testis and pituitary from male Nile tilapia (O. niloticus L.) in the context of social status

Abstract

African cichlids are well established models for studying social hierarchies in teleosts and elucidating the effects social dominance has on gene expression. Ascension in the social hierarchy has been found to increase plasma levels of steroid hormones, follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) as well as gonadosomatic index (GSI). Furthermore, the expression of genes related to gonadotropins and steroidogenesis and signaling along the brain-pituitary-gonad axis (BPG-axis) is affected by changes of an animal's social status. In this study, we use RNA-sequencing to obtain an in-depth look at the transcriptomes of testes and pituitaries from dominant and subordinate male Nile tilapia living in long-term stable social hierarchies. This allows us to draw conclusions about factors along the brain-pituitary-gonad axis that are involved in maintaining dominance over weeks or even months. We identify a number of genes that are differentially regulated between dominant and subordinate males and show that in high-ranking fish this subset of genes is generally upregulated. Genes differentially expressed between the two social groups comprise growth factors, related binding proteins and receptors, components of Wnt-, Tgfβ- and retinoic acid-signaling pathway, gonadotropin signaling and steroidogenesis pathways. The latter is backed up by elevated levels of 11-ketotestosterone, testosterone and estradiol in dominant males. Luteinizing hormone (Lh) is found in higher concentration in the plasma of long-term dominant males than in subordinate animals. Our results both strengthen the existing models and propose new candidates for functional studies to expand our understanding of social phenomena in teleost fish.

Fig 1. Pictures of testes used for RNA-Seq in this study as well as example images for the testis morphology and a fish tank with Nile tilapia in stable hierarchy.

(A), (B) and (C) Pictures of testes from subordinate (S) and dominant (D) males used in this study (according to Table 4). (A) Pictures of family 1 (males IDs 7S, 9S and 8D). (B) Pictures of family 3 (males IDs 30S, 31S and 32D). (C) Pictures of family 2 (males IDs 17S and 18D). (D), (E) Details of testis morphology from a dominant animal (33-month-old male, GSI 0,26; not part of the RNA-Seq experiment) to illustrate morphology of a well-developed testis. (D) Draq5^® staining (cyan) of cell nuclei. A dotted white line labels position of the wall of seminiferous tubules. Asterisks label interstitial compartment including many Leydig cells (show typical cell nuclei with 5μm in diameter). Germ line cells inside of a tubule were labeled according to their developmental stage: A—type A spermatogonia, B—type B spermatogonia, Sc1 –primary spermatocytes, m1 –Sc1 during first meiotic division, Sc2 –secondary spermatocytes, St—spermatids. A white triangle marks the nucleus of selected type A spermatogonia additionally and these triangles were also inserted in (E). The classification of germ line is based on morphological criteria such as cell nucleus diameter and number of cells per cyst. Single cells with largest nuclear diameters of 8 μm or above inside of seminiferous tubules correspond to type A spermatogonia. Further advanced stages (type B spermatogonia and spermatocytes) have a smaller nuclear diameter then type A spermatogonia and show an increasing number of cells with ongoing generations inside of one synchronously developing germ cell cyst. Spermatids can be easily identified by their high number per cyst and their small nuclear diameter. Further details about staging of germ cells and histological methods are described in the Methods section. (E) In addition to the nuclear Draq5^® staining (cyan), Vasa protein caused red fluorescence and Pcna protein caused green fluorescence (most of the green fluorescent cells appear in turquoise because of the overlay with a strong Draq5^® signal) is shown. The undifferentiated type A spermatogonia have the highest levels of the germ line marker Vasa which is reduced during the further differentiation of germ line cells until onset of meiosis. Pcna is expressed in cells at S-phase of the cell cycle. Blood cells (bc) can be identified in the red/green-fluorescence-channel overlay by their typical brownish appearance within the interstitial compartment. (F) Snapshot from a 540 L tank with a long-term stable hierarchy. A dominant white-appearing male holds a territory around the orange tube and at the bottom of the tank. Darker appearing subordinates and females stay outside of its territory. Scale bars in (D) and (E): 20 μm. Diameter of Petri dishes shown in (A)–(C): 90 mm.

Fig 2. Plasma hormone values and TA diameter.

Plasma values of 11-KT, T, E2, Lh and Fsh for each of the experimental animals. Data is represented as obtained by ELISA. Diameter of the TA is also provided for each animal as measured under the confocal microscope. Asteriks (*) marks samples for which hormone measurement was not possible due to limited plasma volume.

Fig 3. Gene expression profiles of testes and pituitaries from dominant and subordinate Nile tilapia show differences between tissues and social groups.

(A) and (B): PCA plots for DEGs in testes (A) and pituitaries (B) show a clear line of division between dominants and subordinate fish. PCAs were computed based on normalized and log-transformed counts of the top 500 variable genes. A second line could be drawn between family 3 (34 months) and the families 1 and 2 (nine to twelve months). Heatmap clustering for the 50 genes with the highest variance in testes (A) and pituitaries (B) based on normalized and log-transformed counts. (C): Numbers of genes sequenced in testes and pituitaries and DEGs used for functional annotation. DEG were defined when the fold change between s-males and d-males had an adjusted p-value of <0.05.

Fig 4. Gene expression in testes and pituitaries of dominant and subordinate Nile tilapia.

Expressions of selected genes as measured by RNA-Seq for testis (A) and pituitary (B) with log2fold change and TPM (transcripts per million). Bold face indicates significant regulation (DEG) with adjusted p-value (pjad) < 0.05. In case the original (Orenil1.0ensembl93) Ensembl gene IDs have retired in the current annotation (O_niloticus_UMD_NMBU), both IDs are listed.

Fig 5. Pathway enrichment with DAVID.

Pathway enrichment analysis results displayed as gene count and fold change. All pathways shown here are significantly enriched in the DEG dataset (Bonferroni corrected p-value <0.05).

Fig 6. RT-qPCR expression of chosen genes from RNA-Seq.

Testis expression of 15 genes measured with RNA-Seq and RT-qPCR of dominant fish compared to subordinate fish. Values are expressed as log2fold change +/- SEM. Significant differences for RT-qPCR data was determined via ANOVA. * = p< 0.05.