Phoenixin-14 alters transcriptome and steroid profiles in female green-spotted puffer (Dichotomyctere nigroviridis)

Abstract

Phoenixin (PNX) is a highly conserved, novel hormone with diverse functions, including hypothalamic control of reproduction, appetite modulation, and regulation of energy metabolism and inflammation. While some functions appear conserved across vertebrates, additional research is required to fully characterize these complex pleiotropic effects. For instance, very little is known about transcriptome level changes associated with PNX exposure, including responses in the hypothalamic-pituitary-gonadal (HPG) axis, which is critical in vertebrate reproduction. In addition, the PNX system may be especially complex in fish, where an additional receptor is likely present in some species. The purpose of this study was to assess hypothalamic and ovarian transcriptomes after PNX-14 administration in female vitellogenic green-spotted puffer (Dichotomyctere nigroviridis). Steroid-related changes were also assessed in the liver and blood plasma. Hypothalamic responses included pro-inflammatory signals such as interleukin 1β, possibly related to gut-brain axis functions, as well as suppression of cell proliferation. Ovarian responses were more widely downregulated across all identified pathways, which may reflect progression to a less transcriptionally active state in oocytes. Both organs shared regulation in transforming growth factor-β and extracellular matrix remodeling (periostin) pathways. Reproductive processes were in general downregulated, but both inhibiting (bone morphogenetic protein 15 and follistatin) and promoting (17-hydroxyprogesterone) factors for oocyte maturation were identified. Select genes involved in reproduction (vitellogenins, estrogen receptors) in the liver were unresponsive to PNX-14 and higher doses may be needed to induce reproductive effects in D. nigroviridis. These results reinforce the complexity of PNX actions in diverse tissues and highlight important roles for this hormone in regulating the immune response, energy metabolism, and cell growth.

Figure 1

Principal component analysis (A) of transcriptomes in ovary control (OC, blue), ovary PNX-14 treated (OT, purple), hypothalamus control (HC, red) and hypothalamus PNX-14 treated (HT, green) samples. (B) Heat map of 36 differentially expressed genes (false discovery rate < 0.05) across both tissues, organized by genes (rows) and samples (columns). Gene names and individual sample IDs are provided on the right and bottom, respectively. Red colors indicate positive log2 fold changes in expression, while blue indicates negative changes. Unknown genes are labeled with their respective gene IDs identified in the Ensembl puffer genome (TETRAODON 8.0). (C) Total differentially expressed genes (P < 0.05) or (D) pathways with percentages (%) in the PNX-14 treatment in ovary (blue) and hypothalamus (red) transcriptomes, with shared entries across both transcriptomes identified in the middle (purple).

Figure 2

RNA-seq normalized count data (first column), relative mRNA expression in qPCR normalized to eef1a (second column), and linear regression analyses (third column) of selected ovary [(A) srl, isr2a, and acta1] and hypothalamus genes [(B) dkk3a, dpydb, and tgfb3]. Each bar represents the mean ± standard error and significance was assessed at P < 0.05. P adjusted (adj.) refers to the multiple test correction in RNA-seq differential expression analyses (false discovery rate < 0.05). For regression analyses, black and gray circles refer to control and PNX-14 treated samples, respectively.

Figure 3

Percent gene sets (%) in the ovary (A) and hypothalamus (B) identified as highly significant (P < 0.01 with > 50% measured entries in a pathway) following PNX treatment were manually assigned functional categories (upper graphs). Numbers above gray bars refer to the total gene set number assigned to each category. Lower graphs identify the percent of gene sets (%) with overall positive (black bars) or negative (striped) median fold changes in each category. P values refer to significant differences from an expected equal frequency of gene sets with positive and negative changes.

Figure 4

Chromosome condensation-associated network identified in the hypothalamus following PNX treatment. Red indicates an upregulated gene in the network (darker shade indicates greater log2 fold change compared to control), while blue indicates a downregulated gene. Shapes and arrows are identified in the legend to the left. This is a proposed mechanism based only on transcriptomic data.

Figure 5

Periostin (postn)-associated network identified in the ovary following PNX treatment. Red indicates an upregulated gene in the network (darker shade indicates greater log2 fold change compared to control), while blue indicates a downregulated gene. Shapes and arrows are identified in the legend to the left. This is a proposed mechanism based only on transcriptomic data.

Figure 6

Relative mRNA expression normalized to eef1a of six steroid-associated liver genes (vtgab, vtgc, shbg, esr1, esr2a, and esr2b) in control and PNX-treated puffers. Each bar represents the mean ± standard error, and significance was assessed at P < 0.05.

Figure 7

Blood plasma concentrations (ng/μl plasma) of six steroids (P4, progesterone; 17OHP, 17-hydroxyprogesterone; CORT, corticosterone; F, cortisol; T, testosterone; 11-KT, 11-ketotestosterone). Each bar represents the mean ± standard error and the asterisk indicates significance (P = 0.049).