Identifying the Interaction of the Brain and the Pituitary in Social – and Reproductive – State of Tilapia by Transcriptome Analyses

Abstract

Introduction: As in all vertebrates, reproduction in fish is regulated by GnRH control on gonadotrophic hormones (GtH) activity. However, the neuroendocrine factors that promote GnRH and GtH activity are unknown. In Nile tilapia (Oreochromis niloticus), sexual activity and reproduction ability depend on social rank; only dominant males and females reproduce. Here, this characteristic of dominant fish allows us to compare brain and pituitary gene expression in animals that do and do not reproduce, aiming to reveal mechanisms that regulate reproduction. Methods: an extensive transcriptome analysis was performed, combining two sets of transcriptomes: a novel whole-brain and pituitary transcriptome of established dominant and subordinate males, together with a cell-specific transcriptome of LH and FSH cells. Pituitary incubation assay validated the direct effect of steroid application on chosen genes and GtH secretion. Results: in most dominant fish, as determined behaviorally, the gonadosomatic index was higher than in subordinate fish, and the leading upregulated pituitary genes were those coding for GtHs. In the brain, various neuropeptide genes, including isotocin, cholecystokinin, and MCH, were upregulated; these may be related to reproductive status through effects on behavior and feeding. In a STRING network analysis combining the two transcriptome sets, brain aromatase, highly expressed in LH cells, is the most central gene with the highest number of connections. In the pituitary incubation assay, testosterone and estradiol increased the secretion of LH and specific gene transcription. Conclusions: the close correlation between behavioral dominance and reproductive capacity in tilapia allows unraveling novel genes that may regulate the HPG axis, highlighting aromatase as the main factor affecting the brain and pituitary in maintaining a sexually active organism.

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“…Four distinct subtypes (SSTR1-3 and SSTR5) and several SSTR isoforms have been characterized in different fish species (reviewed in (70). We identified the receptor types that are expressed in defined pituitary cells by RNA-seq of LH and FSH cell populations separated from the tilapia pituitary (21,71). Three distinct subtypes of SSTRs (SSTR2a, SSTR3a, and SSTR5b) and one isoform (SSTR3b) were found at different levels in these gonadotrophs.…”

“…The RNA-seq library and gene expression were previously published (21,22). To conduct the LH and FSH cell RNA-seq, pituitaries from 20 mature male and 20 mature female transgenic-O.…”

“…We also performed RNA-seq analysis on pituitaries of transgenic tilapia that express GFP in FSH cells and RFP in LH cells (23,24). Of the nine paralogs of SSTR identified in the tilapia genome, only four were expressed in the pituitary (SSTR3b, SSTR5b, SSTR2a, and SSTR3a; marked in blue in Figure 2) (21). Comparison of each receptor expression in LH and FSH cells to their expression in the negative fraction (Figure 3) identified two main SSTRs that had significantly high expression in each gonadotroph: sstr3b was highly expressed in FSH cells, whereas sstr3a and sstr5b were highly expressed in LH cells.…”

“…RNA-seq of FACS-isolated cells identified the different types of SSTRs from pituitaries of transgenic tilapia expressing RFP in LH cells and GFP in FSH cells. Isolated LH cells, FSH cells, and negative pituitary cells were subjected to RNA-seq as described previously (21). The complete transcriptome information is available in GEO through accession number GSE159470.…”

“…We have recently used transgenic tilapia with fluorescent-labeled gonadotrophs to perform RNA-seq on specific populations of LH and FSH cells. Our analysis revealed new candidates, including somatostatin receptors, that may directly regulate these hormones (21). In the present study, we investigated the influence of somatostatin on the regulation of gonadotropins in tilapia.…”