Fishes

17 pages, 4538 KiB

Open AccessArticle

Effects of Maternal Stress on the Development of the Somatotropic Axis During the Larval and Juvenile Stages in Zebrafish (Danio rerio)

by Maira da Silva Rodrigues, Vinícius Prazeres Barbosa Toledo and Rafael Henrique Nóbrega

Fishes 2025, 10(2), 37; https://doi.org/10.3390/fishes10020037 - 21 Jan 2025

Abstract

Stress is recognized as an adaptive response to potentially harmful environmental stimuli. The primary physiological adaptation to stress is an increase in circulating cortisol levels, which, in excess, can be transferred and incorporated into the oocytes of maturing females, affecting the embryonic developmental program. Additionally, maternal energy availability is an essential environmental factor that modulates this program. Based on this background, we investigated the effects of maternal cortisol on the development of the somatotropic axis in zebrafish offspring and juveniles. Zebrafish mothers were divided into two groups based on diet: Group 1 received a cortisol-enriched diet, to mimic maternal stress, while Group 2 (control) received a standard diet, for five days. On the third day after treatment, the control and treated females were bred with untreated males. Offspring were assessed at 0, 24, 48, 72, 96, 120, and 144 h post-fertilization (hpf). Morphological analyses were performed during embryonic development, including survival rate, body length, the presence of pericardial edema, and heartbeat. We examined the gene expression of key somatotropic axis components, including mtor, foxo3a, mafbx, murf1, mstna, gh, igf1, igf2a, igf2b, 11hsdb2, and fkbp5. The study demonstrated that cortisol-treated females significantly influenced offspring development, resulting in higher mortality rates and increased morphological abnormalities, particularly pericardial edema. Gene expression analysis revealed alterations in transcripts related to the somatotropic axis, especially genes involved in protein synthesis, with signs of accelerated growth in the first hour post-fertilization. At 30 days post-fertilization, juveniles from cortisol-treated females displayed a marked increase in muscle bundle size and cross-sectional diameter compared to the control group. Our findings provide valuable insights into the intricate interaction between maternal factors and the development of the somatotropic axis in offspring. Full article

(This article belongs to the Section Physiology and Biochemistry)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Experimental design. (1) Two groups of female zebrafish (n = 30) were assigned into three replicates per experimental group. One group was fed commercial food, while the other one was fed commercial food containing 0.5 mg per feed of hydrocortisone. Zebrafish mothers received approximately 25 µg cortisol/g body mass. Fish were fed twice per day for 5 days. (2) On day 3 post-treatment, five females from each group were transferred to breeding tanks with untreated males. (3) Fertilized eggs were collected in Petri dishes containing embryo medium. (4) Eggs, embryos, and larvae were collected at 0 to 144 hpf for survival rate and phenotype characterization, such as length and pericardial edema. The eggs, embryos, and early larvae from 0 to 72 hpf were counted and pools were collected for gene expression analysis. Embryos at 48 hpf from the control and cortisol groups were taken to calculate heartbeat rate per minute (n ~ 20/condition). (5) Juveniles at 30 dpf were also collected for the histological analysis of muscle fiber in both groups. The illustration is available online: <a href="https://BioRender.com" target="_blank">https://BioRender.com</a> (accessed on 1 November 2024). Full article ">Figure 2
Somatotropic and HPI-related genes in embryos and early larvae at 0 to 72 hpf from mothers fed with a hydrocortisone enriched diet (0.5 mg per g feed) compared to the untreated group (control) (dotted black line set at 1). The selected genes mtor (mammalian target of rapamycin) (A); foxo3a (forkhead box O3) (B); mafbx (muscle atrophy F-box) (C); murf1 (muscle ring-finger protein-1) (D); mstna (myostatin) (E); gh (growth hormone) (F); igf1 (insulin-like growth factor 1) (G); igf2a (insulin-like growth factor 2a) (H); igf2b (insulin-like growth factor 2b) (I); fkbp5 (glucocorticoid receptor chaperone protein) (J); and 11hsdb2 (hydroxysteroid 11-beta dehydrogenase 2) (K) were evaluated. Ct values were normalized with β-actin and expressed as relative values of the control (untreated fish) levels of expression. Bars represent the mean ± SEM fold change (n = 5 pools of 20 individuals for each pool) relative to the control. Student’s unpaired t-test, * p < 0.05, ** p < 0.01, *** p < 0.001. Full article ">Figure 3
The survival rate of embryos and larvae at 0 to 144 hpf (A) was demonstrated in both control and treated animals. Embryo and larvae length at 48 to 72 hpf (B) were measured (bars represent the mean ± SEM; ANOVA followed by Tukey’s multiple comparison tests or Student’s unpaired t-test). Distinct letters denote significant differences; p ≤ 0.05). The phenotype of embryos and early larvae from control females (C,D) was compared with those of progeny impacted by maternal transfer of corticosteroid (E,F). Control animals showed normal pericardium morphology (C,D). However, embryos from cortisol-treated females were affected. Arrows highlight the presence of pericardial edema (E,F). Scale bar = 500 µm (n = 30, basal and treatment). Full article ">Figure 4
The heartbeats of embryos at 48 hpf for the control (red circles) (n = 12) and maternal cortisol (blue circles) (n = 18) groups were demonstrated. No significant heartbeat difference was found between the control and treatment embryos. Student’s unpaired t-test, p = 0.1459. Full article ">Figure 5
Longitudinal histological sections of muscle from 30 dpf zebrafish juveniles from females fed with a commercial diet (A) or a diet supplemented with cortisol (B). Muscle bundle lengths at 30 dpf for juveniles from control (black) and cortisol-treated (red) females (C). (Bars sampled in each group represent the mean ± SEM; Student’s unpaired t-test, * p < 0.05). Scale bar = 100 µm. Staining: Toluidine blue (n = 3, basal and treatment). Full article ">Figure 6
Histological cross-sections of muscle in zebrafish juveniles at 30 dpf from females fed a commercial diet (control) (A,C) or a diet supplemented with cortisol (B,D). The cross-sectional diameters of muscle fibers in both progeny from non-treated and cortisol-treated females were measured in offspring at 30 dpf (E). The proportion of thin (<9 µm) and thick (>9 µm) muscle fibers in zebrafish juveniles at 30 dpf from control and cortisol-treated females were measured (F). (Symbols or bars represent the mean ± SEM; Student’s unpaired t-test, * p < 0.05; *** p < 0.001). Scale bar = 25 µm (A,B); 10 µm (C,D). Staining: Toluidine blue (n = 5; 75 muscle fibers per animal, basal and treatment). Full article ">

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