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. 2024 Jul 18;45(4):791–804. doi: 10.24272/j.issn.2095-8137.2023.369

Metabolomic-based analysis reveals bile acid-mediated ovarian failure induced by low temperature in zebrafish

Wen-Hao Li 1,2,3,#, Zhi-Qiang Li 1,3,#, Meng-Di Bu 1,3,#, Jia-Zhen Li 1,3, Liang-Biao Chen 1,3,*
PMCID: PMC11298673  PMID: 38894522

Abstract

As ectotherms, fish are highly sensitive to temperature fluctuations, which can profoundly impact their reproductive cycles. In this study, we investigated the fertility and histological characteristics of zebrafish (Danio rerio) ovaries exposed to a temperature gradient ranging from the thermopreferendum temperature of the species, 27°C, to lower temperatures of 22°C, 20°C, and 13°C over a period of two weeks. Comparative metabolomic (six biological replicates for each temperature) and transcriptomic (four biological replicates for each temperature) analyses were conducted under the four temperature conditions. Results indicated that lower temperatures inhibited oocyte development and differential metabolites were involved in steroid hormone production, antioxidant function, and lipid and protein catabolism. Disrupted reproductive hormones, increased proteolysis, and lipid degradation significantly impeded oocyte development and egg maturation. Notably, a significant increase in bile acid content was noted in the ovaries of the cold-treated fish, indicating that bile acids play a critical role in ovarian failure. Overall, these findings provide valuable insights into the mechanisms governing the reproductive response of fish to cold stress.

Keywords: Cold stress, Ovarian failure, Metabolome, Zebrafish

INTRODUCTION

Reproductive performance in fish is closely associated with the economic outcomes of aquaculture (Gjerde, 1986). Cold temperatures present a major challenge in completing the reproductive cycles for many farmed species, such as tilapia (Oreochromis niloticus) and largemouth bass (Micropterus salmoides). The inhibition of reproduction due to cold temperatures not only prolongs the farming period but also negatively impacts the quality and quantity of spawned eggs, leading to higher larval mortality and lower productivity (El-Naggar et al., 2000; Islam et al., 2022). Studying how the reproductive system responds to cold temperatures provides crucial biological insights for enhancing reproductive performance in fish aquaculture. Temperature strongly influences animal survival and reproduction (Alix et al., 2020; Ou et al., 2018), particularly in poikilothermic animals such as fish (Dahlke et al., 2020; Hu et al., 2015). As fish cannot regulate their body temperature internally, they are heavily reliant on their surrounding environment for thermoregulation (Lushchak, 2011). Genomic (Chen et al., 2008, 2019), transcriptomic (Chen et al., 2008; Hu et al., 2015, 2016; Long et al., 2013; Windisch et al., 2011), and epigenomic (Campos et al., 2013; Han et al., 2016) analyses have been instrumental in unraveling the genetic networks associated with the response of fish to cold stress. Prior studies on cold-induced apoptosis in fish have shown that different tissues respond variably to cold stress, with gill and kidney tissues exhibiting higher sensitivity to critical thermal limits (Hu et al., 2016; Wang et al., 2023). When ambient temperatures drop below the preferred thermal range for fish, female gamete production may be inhibited. Spawning adults, like developing embryos, represent the most temperature-sensitive life stage (Dahlke et al., 2020), with thermal fertility limits (TFLs) typically preceding critical thermal limits (CTLs) (Parratt et al., 2021). This can result in significant damage to the ovaries, leading to infertility in many species, even if other tissues are not notably affected (Servili et al., 2020). Even mild non-life-threatening temperature changes can disturb fish reproduction (Dahlke et al., 2020).

As fish evolved and underwent adaptive radiation, they expanded from equatorial to polar regions, with each species developing optimal reproductive temperatures (Donelson et al., 2010; Kock, 1992; Lopes et al., 2020). The thermal sensitivity of fish ovaries varies markedly among tropical, temperate, and polar species (Van Der Kraak & Pankhurst, 1997). At their optimal temperatures, vertebrates maintain body temperatures that closely align with their metabolic and reproductive optima (Kennedy & Mihursky, 1967). In adverse conditions, organisms can adapt through their stress response system, which comprises a set of physiological responses enabling survival while sacrificing energy-intensive functions such as reproduction (Barton, 2002). Cold stress (18°C or 10°C for 48h) results in the dysregulation of multiple genes associated with reproduction in female zebrafish, leading to ovarian damage (Zhao et al., 2023). However, the physiological mechanisms underlying the effects of "cold noxious" temperatures on fish ovaries, especially in gradient cold temperatures, remains largely unknown.

Metabolomic adjustments are fundamental for fish adaptation to environmental stress, primarily attributable to stress-induced coordinated genetic and epigenetic reprograming (Liu et al., 2022b). Previous gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS)-based metabolomic analyses found that metabolites involved in energy metabolism and basic amino acids increased significantly in black rockfish (Sebastes schlegelii) exposed to acute high thermal stress (27°C for 12 h), while certain fatty acids increased after acute cold stress (5°C for 12 h) (Song et al., 2019). Another study using GC-MS analyzed the serum metabolic responses of Nile tilapia (Oreochromis niloticus) subjected to Streptococcus agalactiae infection at normal (25°C) and high (35°C) temperatures and found that glycolysis was inhibited and arachidonic acid consumption may be associated with mortality following high-temperature infection (Hu et al., 2020). To date, however, research on the impact of cold temperatures on metabolomic changes in the reproductive organs of fish remains limited.

The aim of this study was to identify metabolite biomarkers that respond to cold stress and assess the impact of cold stress on fish reproduction. The female reproductive system was selected to investigate ovarian impairment following 2 weeks of exposure to a cold-stress gradient, measuring fertility and histological changes. Reproductive metabolic changes and adaptations to cold were assessed in the ovaries of cold-exposed fish using a global metabolomics approach based on liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified several cold-responsive biomolecules and biomarkers in the ovary and revealed bile acid-mediated ovarian failure in cold stress.

MATERIALS AND METHODS

Low temperature treatment and sample preparation

The optimal temperature range for zebrafish is 26°C to 28°C (López-Olmeda & Sánchez-Vázquez, 2011), so we selected 27°C as the control group. The temperature range for zebrafish embryo development is 22°C to 34°C (Urushibata et al., 2021), so we chose 22°C as the first treatment temperature. The critical temperature determining the survival of embryonic development is 20°C (Schnurr et al., 2014), which we selected as the second treatment temperature. The critical temperature resulting in the death of most zebrafish within 24 h is 12°C (Wu et al., 2015), thus we adopted a sublethal temperature (13°C) as the third treatment temperature. After treating the zebrafish at the four different temperatures for 7 days, the histological changes were not pronounced. However, distinct phenotypic changes were observed after 14 days and a significantly reduced number of mature follicles were noted by day 30 (Supplementary Figure S1). As our objective was to observe the process of ovarian failure, we opted for a treatment duration of 14 days.

Wild-type (WT) AB strain zebrafish breeding was conducted in the laboratory of Shanghai Ocean University. Sexually mature female zebrafish (7–8 months old), cultured under normal temperatures (27°C), were transferred to tanks with four separate flow circulation systems, where water temperature could be adjusted. The selected zebrafish were uniform in weight and size. Starting at 27°C, the water temperature in three tanks was gradually cooled to 22°C, 20°C, and 13°C respectively, at a rate of 0.5°C/h, while one tank remained at 27°C. The flow circulation systems were then activated at the set temperatures, and the fish were exposed to these temperatures for 14 days under a light/dark cycle of 14 h/10 h. The four sets of fish were fed with freshly hatched Artemia twice daily (0900h and 1800h). Six biological replicates were established for each temperature, with each replicate containing five fish. Fish from each temperature treatment were sampled for ovarian imaging, histological analysis, and LC-MS/MS measurement. All animal experiments adhered to the guidelines of, and were approved by, the Animal Research and Ethics Committees of Shanghai Ocean University (SHOU-DW-2021-061).

Histological analysis

The ovaries were collected and immersed in 4% paraformaldehyde (Sangon Biotech, China) overnight, with subsequent dehydration through a serial gradient of alcohol (Sangon Biotech, China) at 50%, 70%, 80%, 90%, and 100% (1 h each). The dehydrated ovaries were transferred to lemosol (Wako, Japan) to increase transparency, then immersed in paraffin for 2 h and embedded in wax blocks. The samples were sliced into 5 μm sections using a Leica microtome (Leica biosystems, Germany), dried overnight at 37°C, dewaxed in xylene, rehydrated in decreasing concentrations of ethanol (Sangon Biotech, China), washed in distilled water, and stained with hematoxylin-eosin (H&E, Beyotime, China). The sections were then dehydrated under increasing concentrations of ethanol and xylene (Sangon Biotech, China), dried in a fume hood, and mounted on glass slides with a neutral balsam (Sangon Biotech, China) mounting medium for photography using a microscope (Nikon, Japan).

Ultra-high performance (UHP) LC-MS/MS metabolomic analysis

Fresh ovarian tissues were rapidly frozen in liquid nitrogen and stored at –80°C. Subsequently, 100 mg of each tissue sample was homogenized in liquid nitrogen, with the resulting homogenate resuspended with pre-chilled 80% methanol (ThermoFisher, USA) containing 0.1% formic acid (ThermoFisher, USA) through vigorous vortexing. The samples were then incubated on ice for 5 min and centrifuged at 15 000 ×g and 4°C for 20 min. The supernatant (400 μL) was further diluted to a final concentration of 53% methanol using LC-MS grade water (Merck, Germany). The resulting samples were transferred to fresh Eppendorf tubes and subjected to centrifugation step at 15 000 ×g and 4°C for 20 min. Finally, the supernatant was injected into the LC-MS/MS system for analysis. Quality control (QC) samples were established by taking 10 μL of each experimental sample and mixing it evenly.

The raw data files produced by UHPLC-MS/MS were processed using Compound Discoverer v.3.1 (CD3.1, ThermoFisher, USA) to execute peak alignment, peak picking, and quantitation for each metabolite. Key parameters were configured as follows: retention time tolerance of 0.2 min; actual mass tolerance of 5 ppm; signal intensity tolerance of 30%; signal/noise ratio of 3; and minimum intensity. Following this, peak intensities were normalized to total spectral intensity. The normalized data were utilized to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Subsequently, peaks were matched with the mzCloud (https://www.mzcloud.org/), mzVault, and MassList databases to acquire accurate qualitative and relative quantitative results. Statistical analyses were conducted using R statistical software (v.3.4.3) and Python (v.3.5.0). In cases where data deviated from normal distribution, attempts at normalization were made using the area normalization method: metabolite original quantitative value/(sum of metabolite quantitative values in sample/sum of metabolite quantitative values in QC1).

After metabolomic data transformation using metaX package in R, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed. Metabolites with coefficients of variance (CV) less than 0.3 were retained through filtering. Three sets of differential metabolite data, namely 27°C vs. 22°C, 27°C vs. 20°C, and 27°C vs. 13°C, were screened using the NovoMagic platform (https://magic.novogene.com/). Differential metabolites (DMs) were screened based on a variable importance for projection (VIP) score>1, P<0.05, and fold-change>1.5 or <0.67. Metabolites were annotated using the Kyoto Encyclopedia of Genes and Genomes database (KEGG; https://www.genome.jp/kegg/pathway.html), Human Metabolome Database (HMDB; https://hmdb.ca/metabolites), and LIPID MAPS database (https://lipidmaps.org).

Metabolite enrichment

The KEGG database in MetaboAnalyst (https://www.metaboanalyst.ca/MetaboAnalyst/upload/EnrichUploadView.xhtml) was used to perform enrichment analyses of all differential metabolites obtained from LC-MS/MS. Subsequently, the ggplot2 package in R was used to generate a bubble plot for metabolic pathways with P<0.05.

Soft clustering analysis

Clustering analysis was conducted to investigate differentially expressed metabolites using the Mfuzz package. The Fuzzy C-Means (FCM) clustering algorithm, a component of the package, employs soft partition clustering, which requires two primary parameters (c=number of clusters, m=fuzzification parameter). The iterative refinement procedure was used to determine the optimal values of c and m, calculated to be 16 and Mestimate (data), respectively.

Transcriptome analysis

Total RNA was extracted using TRIzol reagent (Sigma-Aldrich, USA). Strand-specific RNA-seq libraries were prepared using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina v.2 (Vazyme Biotech, China) and subjected to 150 bp paired-end sequencing on the Illumina NovaSeq 6000 platform (USA). Trimmomatic (v.0.36) was employed to remove low-quality reads and sequencing adapters, followed by clean read alignment to the zebrafish reference genome GRCz11 (v.106) using STAR (v.2.7.10a). Read counts for each gene were calculated using featureCounts (v.2.0.1). Finally, fragments per kilobase million (FPKM) values for each gene were obtained using DESeq2.

Correlation analysis of transcriptomic and metabolomic data

To assess the concordance of changes between the transcriptome and metabolome, Pearson correlation analysis was performed between differentially expressed genes in the transcriptome and metabolites in the metabolome. First, genes with significant changes from the transcriptome data were identified. Subsequently, Pearson correlation coefficients and P-values between the selected genes and target metabolites were calculated. The Benjamini-Hochberg method was applied for multiple testing correction using the P-adjust function in R to control the false discovery rate (FDR). Genes with adjusted P-values less than 0.05 and functionally relevant to the target metabolites were selected, and Pearson correlation coefficients were used for visualization. The corrplot package in R was used to present the correlation plots.

Glycodeoxycholic acid (GDCA) treatment

Adult female zebrafish (7–8 months old), without any abnormal temperature treatment, were maintained at 27°C in tanks with 10 L of filtered water. Subsequently, 40 mg of GDCA hydrate (Xianding Biotechnology, China, B-HI304) dissolved in 400 μL of dimethyl sulfoxide (DMSO, Sangon Biotech, China) was added to 1 L of water (40 mg/L), while 400 μL of DMSO without GDCA was added to 1 L of water for the control group. The selection of 40 mg/L GDCA for zebrafish treatment was based on assays showing that total bile acids can reach 20–25 mg/L in human plasma under bile metabolic disorders (Roy-Chowdhury et al., 2020) and serum bile acid content can exceed 100 mg/L in cases of obstructive jaundice (Song & Kew, 1983). In addition, previous experiments have demonstrated no negative impact on zebrafish survival at a concentration of 40 mg/L GDCA (Liu et al., 2022a). During GDCA treatment, half of the water was replaced with fresh, filtered water daily. The diel rhythm, temperature, and GDCA concentration were maintained for 14 days. Three replicates, each containing 10 fish, were conducted.

RESULTS

Low temperature disrupted oocyte development

Adult female zebrafish were exposed to four different temperatures for two weeks, representing normal (27°C), mild (22°C and 20°C), and severe (13°C) cold stress conditions. Cold stress altered ovarian histology, as revealed by H&E staining of ovarian sections (Figure 1). At 27°C, the ovaries were well-developed, with a high proportion of mature oocytes and yolk-filled eggs. A similar histological appearance was found in the ovaries of fish acclimated to 22°C, with a slightly reduced percentage of maturing eggs and overall ovary size compared to the normal group. However, when the treatment temperature decreased to 20°C, the percentage of immature oocytes increased significantly, worsening under 13°C treatment. These results indicated that consistent cold stress compromised oocyte maturation by disrupting oocyte development. More pronounced phenotypes were achieved under 30 days of 13°C treatment, with oocyte development primarily halting at the early vitellogenic stage, and mid-vitellogenic oocytes shrinking (Supplementary Figure S1). Severe cold exposure resulted in defects in oocyte development and oogenesis in zebrafish.

Figure 1.

Figure 1

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Morphology of ovaries and eggs in zebrafish acclimated to different cold temperatures

Zebrafish and their ovaries were examined after 14 days of exposure to four different temperature treatments. A: Appearance of zebrafish exposed to four different temperatures. B: Morphological appearance of ovaries from fish exposed to different temperatures. C: Central region of ovary was extracted using forceps and photographed under a ZEISS microscope (ZEISS, Germany) (12.5×). D, E: Retrieved ovaries were dispersed using a pipette and imaged under the same ZEISS microscope (10× and 25×). F: H&E-stained ovarian sections, photographed under a Nikon microscope (Nikon, Japan) (40×). FG: Fully-grown stage.

Metabolite profiling in ovaries exposed at different temperatures

UHPLC-MS/MS was conducted to elucidate the metabolomic signature of ovaries exposed to cold stress. A total of 1 605 metabolites were identified in the ovaries of zebrafish from the four different temperature groups (Supplementary Tables S1, S2). Correlation analyses of the six QC samples based on total ion chromatograms (TIC) demonstrated high retention time and response intensity of the mass spectral peaks, indicating high stability and repeatability (Supplementary Figure S2). The PLS-DA results showed a clear separation of the 27°C group from the 13°C, 20°C, and 22°C groups, respectively. The 20°C group profile was more similar to that of the 22°C group than to the other groups (Figure 2A). These results suggested that cold temperatures exerted a significant impact on the metabolic profiles of the zebrafish ovary.

Figure 2.

Figure 2

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Metabolite screening

A: PLS-DA was performed on ovarian metabolome of zebrafish treated for 14 days at 27°C, 22°C, 20°C, and 13°C. B: Venn diagram of up-regulated or down-regulated metabolites in 13°C vs. 27°C, 20°C vs. 27°C, and 22°C vs. 27°C comparisons and their overlap. In total, 101 up-regulated and 49 down-regulated metabolites were identified.

In total, 451, 449, and 515 differential metabolites were identified in the 22°C vs. 27°C, 20°C vs. 27°C, and 13°C vs. 27°C comparisons, respectively (Supplementary Table S3). Venn plot analysis revealed 101 up-regulated and 49 down-regulated metabolites common to all three comparisons (Figure 2B). The up-regulated differential metabolites were enriched in androgens, bile acids, and protein derived metabolites (i.e., amino acids and dipeptides), while the down-regulated differential metabolites were enriched in molecular functions such as antioxidants and the neuroendocrine factor epinephrine (Supplementary Table S4). Furthermore, specifically increased metabolites in the 13°C group revealed that several amino acids were significantly up-regulated at this temperature (Supplementary Figure S3).

KEGG enrichment analysis identified 15 differential metabolic pathways (Supplementary Figure S4), including pathways related to antioxidant metabolism (e.g., glutathione metabolism), steroid metabolism (e.g., steroid hormone biosynthesis), and amino acid metabolism pathways (e.g., arginine biosynthesis and glycine, serine, and threonine metabolism).

Utilizing the Mfuzz R package, metabolites were clustered to reflect change trends with decreasing temperatures, yielding 16 clusters (Figure 3). Clusters 2, 4, 10, and 14 exhibited the opposite trend of change with decreasing treatment temperature (Figure 3A), while clusters 1, 5, 9, and 16 showed a gradual decreasing trend (Figure 3B), and the remaining clusters displayed irregular patterns of change (Figure 3C).

Figure 3.

Figure 3

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Cluster analysis of metabolites

A: Metabolites were clustered using Mfuzz based on changes with decreasing temperature, resulting in 16 clusters. Clusters 2, 4, 10, and 14 exhibited a gradual increase in metabolites with decreasing temperature. B: Clusters 1, 5, 9, and 16 exhibited a gradual decrease in metabolites with decreasing temperature. C: Remaining clusters exhibited irregular variations with temperatures.

Effects of cold stress on steroid hormones and prostaglandins (PGs)

Tracing the biochemical pathways for synthesizing the reproductive steroid hormones, we found significant alteration of several metabolites after cold treatment (Figure 4A). Among these metabolites, pregnenolone, 5β-androstane-3,17-dione, and testosterone were generally increased with the decreasing of the exposure temperatures. However, no significant changes were found in the amount of estrone and estradiol (E2) albeit the upregulation of its precursor 5β-androstane-3,17-dione, suggesting a biased increase of male hormone testosterone in the cold stressed ovaries. Besides the upregulation of the male hormones, four types of PGs were steadily increased in the cold exposed ovaries (Figure 4B).

Figure 4.

Figure 4

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Intermediate product of estradiol synthesis and up-regulated PGs

A: Flowchart shows estradiol synthesis from cholesterol, with enzymes in yellow rectangles (Javitt et al., 2001; Nerusu et al., 2017; Stocco, 2000; Swart et al., 2003). Bar charts next to metabolites show changes in zebrafish ovaries post low-temperature treatment. X-axis is temperature (°C), y-axis is metabolite levels. Red-shaded areas indicate hormone up-regulation, blue-shaded areas indicate down-regulation under low temperatures. Asterisks denote significance (*: P<0.05; **: P<0.01; ***: P<0.001). Hormone molecular formulas were from http://www.chemspider.com. B: Levels of four prostaglandins (PGF2β, PGE2, PG, and PGA2) in cold-acclimated ovaries.

Reduced anti-oxidants and increased protein and triglyceride degradation products

A marked decrease was found in several compounds involved in oxidative stress, including S-(methyl)glutathione, L-ascorbate, reduced L-glutathione (GSH), and oxidized L-glutathione (GSSG) (Figure 5A), suggesting a reduction in anti-oxidative capacity in cold exposed ovaries. Proteolysis also increased, as evidenced from the accumulation of various dipeptides and free amino acids, such as N-glycyl-L-proline, Gly-Val, H-Gly-Pro-OH, L-alanyl-L-proline, Val-Ser, glycyl-proline, leucyl-proline, prolyl-leucine, L-threonine, L-lysine, threonine, DL-arginine, and L-cystine (Figure 5A). Similarly, a steady increase in various long-chain polyunsaturated fatty acids (LPUFAs) was detected, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and arachidonic acid (ARA) (Figure 5A). Various fatty acyl esters of hydroxy fatty acids (FAHFAs) were also identified, which can enhance insulin-stimulated glucose transport and help meet energy demands (Tan et al., 2019; Yore et al., 2014) (Supplementary Figure S5). Through combined transcriptomic and metabolomic analysis, we identified genes associated with protein metabolism (Figure 5B). As anticipated, genes involved in protein degradation showed a positive correlation with up-regulated amino acids, and their expression levels significantly increased, including proteasome (psm) and cacul1 (Supplementary Figure S6). Conversely, genes involved in protein synthesis exhibited a negative correlation with up-regulated amino acids, and their expression levels were significantly decreased, including kri1, mrps34, and mtg2 (Supplementary Figure S6). These findings indicate a shift from protein synthesis to protein catabolism in zebrafish ovaries exposed to low temperatures.

Figure 5.

Figure 5

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Down-regulated antioxidants, up-regulated amino acids, LPUFAs, and Pearson correlation analysis

A: As temperature decreased from 27°C to 13°C, levels of four antioxidants declined in zebrafish ovaries, while multiple amino acids and various LPUFAs showed an up-regulation. Numerical values of metabolite levels were standardized. B: Pearson correlation between up-regulated amino acids and genes associated with protein metabolism.

Accumulation of bile acids in the ovary

With the decrease in treatment temperature, there was a general increasing trend in several bile acids and their derivatives, including GDCA (hydrate), taurolithocholic acid 3-sulfate, lithocholic acid, tauroursodeoxycholic acid dihydrate, and taurolithocholic acid sodium salt (Figure 6). All five bile acids, derived from cholesterol, facilitate fat and vitamin absorption (Smith et al., 2009).

Figure 6.

Figure 6

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Substances involved in bile acid formation at low temperature

Levels of five bile acids formed indirectly through cholesterol metabolism were significantly elevated under low temperatures. X-axis represents temperature (°C), y-axis represents metabolite levels. Asterisks indicate significant differences (*: P<0.05; **: P<0.01; ***: P<0.001). Normalization process: metabolite original quantitative value/(sum of metabolite quantitative values in sample/sum of metabolite quantitative values in QC1).

Bile acid treatment caused ovarian degradation in zebrafish

To test whether an increase in bile acids plays a role in ovarian failure, we treated adult female zebrafish with GDCA, which showed a steady and largest increase in the low temperature-exposed ovaries. At a final concentration of 40 mg/L, GDCA was used to treat aged-matched adult female zebrafish derived from the same parent with temperatures maintained at 27°C for both the GDCA-treated and untreated groups (Figure 7A).

Figure 7.

Figure 7

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Comparation between control and GDCA-treated groups

A: Experimental set up. B: Bar graph shows GSI. C: Proportions of oocytes in five developmental stages. D: From top to bottom: external appearance of zebrafish, appearance of ovary after abdominal dissection, partial ovary, dispersed ovary, enlarged dispersed imaged under ZEISS microscope (25×), H&E-stained ovary paraffin section, imaged using a Nikon microscope (40×). FG: Fully-grown stage; MV: Mid-vitellogenic stage; EV: Early vitellogenic stage; PV: Previtellogenic stage; PG: Primary growth stage.

After 14 days of treatment, ovarian weight, body weight, and the gonadosomatic index (GSI) were calculated (Supplementary Table S5). Results indicated that GSI was significantly lower in the GDCA-treated group than in the control group, suggesting a significant adverse effect of GDCA on ovarian development (Figure 7B). Histological analysis of the GDCA-treated zebrafish ovaries revealed no significant changes in the proportions of oocytes at the primary growth (PG) and mid-vitellogenic (MV) stages. However, there was a significant increase in oocytes at the previtellogenic (PV) and early vitellogenic (EV) stages, while the proportion of fully grown (FG) stage (mature) oocytes decreased by 11% compared to the control group (Figure 7C). The GDCA-treated ovaries showed atrophy (Figure 7D; Supplementary Figures S7, S8). These results suggest that a bile acid increase inhibits oocyte development at the PV and EV stages, while simultaneously inhibiting yolk accumulation (Figure 7).

DISCUSSION

Reproductive capacity of female fish is limited by cold stress

At present, there is a limited understanding of the molecular mechanisms underlying the impact of reduced temperatures on ovarian development in fish. Exploring this phenomenon could provide novel insights into fish reproduction under low-temperature challenges. In our investigation, we initially employed H&E staining to illustrate that exposure to low temperatures resulted in a reduction in zebrafish ovary size, suppression of oocyte development, and a decline in the mature oocyte population (Figure 8A). We also explored the mechanisms underlying the inhibition of zebrafish oocyte development at low temperatures using untargeted metabolomic analyses. Our findings revealed that cold stress perturbed sex hormones, altered both protein and fatty acid metabolism, reduced antioxidant levels, and increased bile acid content. These metabolic changes synergistically contributed to ovarian failure in zebrafish under cold stress (Figure 8B).

Figure 8.

Figure 8

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Model diagram of ovarian abortion in zebrafish induced by low temperature

A: Conditions of subnormal rearing temperature inhibited the growth and development of zebrafish oocytes. Decrease in temperature led to a gradual reduction in number of mature oocytes. B: Left side shows steps involved in synthesis of estradiol from cholesterol. Right side shows bile acids entering oocyte, leading to the breakdown of yolk granules into amino acids and long-chain polyunsaturated fatty acids (LPUFAs), ultimately inhibiting further oocyte maturation.

Cold treatment up-regulates amino acids, LPUFAs, and FAHFAs

The psm family ring finger protein 11a (rnf11a) (Chen et al., 2011; Joazeiro & Weissman, 2000), CDK2-associated cullin domain 1 (cacul1), and NEDD8 activating enzyme E1 (nae1) (Chiba & Tanaka, 2004) regulate protein degradation as components of ubiquitin ligases. Conversely, the loss of kri1l (Jia et al., 2015), mitochondrial ribosomal proteins (MRPS), and mitochondrial ribosome-associated GTPase 2 (mtg2) (Miller et al., 2004) impairs protein synthesis. In our study, low temperatures led to the up-regulation of genes involved in protein degradation (psm family and cacul1) and the down-regulation of genes involved in protein synthesis (kri1l, mrps34 and mtg2), ultimately leading to an increase in free amino acid levels in the ovaries under cold treatment.

Our data indicated that cold treatment significantly increased the accumulation of LPUFAs and FAHFAs in the ovary. Recent research has shown that endogenously synthesized DHA promotes pregnenolone production by regulating the transcription of cyp11a1, thereby enhancing oocyte maturation and quality (Li et al., 2024). As an LPUFA, EPA plays an important role in various organisms, from microorganisms to mammals, by maintaining plasma membrane fluidity and facilitating cell division (Andersson et al., 2007; Kawamoto et al., 2009; Sato et al., 2008). Bangia fuscopurpurea cultured at 4°C shows a 29.8% increase in EPA content, along with an up-regulation in desaturase and elongase genes associated with EPA biosynthesis, which contributes to improved membrane fluidity and cold tolerance (Cao et al., 2017). ARA has been shown to induce oocyte maturation (Jiang et al., 2019), respond to cold stress (Song et al., 2019), and inhibit activation of channel transient receptor potential (melastatin)-8 (TRPM8) (Andersson et al., 2007). Additionally, ARA increases in the ovary, which may serve to maintain stability of the immune system to cope with prolonged cold stress (Qi et al., 2022). It is hypothesized that low temperatures lead to the conversion of lipids into LPUFAs and FAHFAs, providing energy for zebrafish to withstand the cold (Tan et al., 2011; Wood, 2020), consistent with the increase in LPUFA content in Nile tilapia exposed to suboptimal cold conditions (Corrêa et al., 2023).

We speculate that the degradation of proteins and fatty acids may contribute to ovarian decline. Previous research has shown that disruption of lipid homeostasis in zebrafish is detrimental to reproductive development (Xu et al., 2023). Temperature stress induces the redistribution of protein and lipid metabolism from anabolism toward catabolism, strongly impeding growth and reproduction (Iwama et al., 1997; Schreck & Tort, 2016). Under long-term cold treatment, metabolic energy is likely redirected towards cold resistance at the expense of reproduction, resulting in ovarian shrinkage.

Cold treatment reduces antioxidant levels

Previous studies have shown that marked reductions in temperature can elevate antioxidative compound levels in various plants and animals (Lubkowska et al., 2019; Proietti et al., 2009; Wang et al., 2022b). However, changes in antioxidant levels depend on the duration of cold treatment. For example, Wang et al. (2022a) observed a sharp increase in antioxidant enzymes, including GSH, in zebrafish embryonic fibroblast (ZF4) cells after 2 days of cold stress, but a significant decrease in antioxidant enzymes compared to the control group after three months of cold acclimation. And, we observed a decrease in both GSH and GSSG levels during cold treatment (Figure 5A), similar to findings in the liver of the high-altitude frog Nanorana pleskei (Zhang et al., 2021). Cold exposure in juvenile Chinese soft-shelled turtles (Pelodiscus sinensis) leads to a decrease in brain ascorbate concentration (Chen et al., 2015), while the brain levels of ascorbic acid and GSH are lower in winter than in summer in Trachemys scripta elegans turtles (Pérez-Pinzón & Rice, 1995). Our study revealed a decline in GSH and ascorbic acid levels in the zebrafish ovary. We postulate two potential causes for the decline: First, low temperatures may hinder ovarian development, increasing the proportion of immature oocytes, which generally exhibit lower concentrations of GSH and ascorbic acid compared to mature oocytes (Funahashi et al., 1994). Second, the ovary may require greater consumption of antioxidant molecules to neutralize reactive oxygen species (ROS) and maintain oocyte redox status under cold exposure. Consequently, GSH and ascorbic acid levels decrease in response to the increased demand for antioxidant defense. These factors may act synergistically to reduce GSH and ascorbic acid levels in the zebrafish ovary under cold treatment.

The down-regulation of antioxidants can lead to an overproduction of ROS. Oxidative stress negatively impacts the reproductive system and can even lead to pathological conditions (Agarwal et al., 2006). The pathological consequences of a decreased antioxidant defense system include reproductive disorders such as polycystic ovary syndrome (PCOS) and infertility (Ruder et al., 2008; Sabuncu et al., 2001). Similar to our findings, a significant increase in atretic antral follicles in rat ovaries occurs after blocking GSH synthesis with the inhibitor buthionine sulfoximine (Lopez & Luderer, 2004). Therefore, the down-regulation of antioxidants under low temperatures may be a significant contributor to ovarian failure in zebrafish.

Cold stress up-regulates cholesterol metabolism, altering profile of steroid hormone and accumulation of bile acids

Testosterone is synthesized from cholesterol through a process catalyzed by cholesterol side-chain cleavage enzymes (CYP11a), producing pregnenolone. Pregnenolone is then converted into testosterone through a series of enzymatic reactions involving 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17), and 17β-hydroxysteroid dehydrogenase (17β-HSD) (Figure 4A). High concentrations of testosterone can impede follicular development, resulting in reduced reproductive capacity (Beloosesky et al., 2004; Liu et al., 2015, 2021; Yu et al., 2020). Additionally, the accumulation of PGs may correspond to an increase in undeveloped eggs under cold stress, as oocytes failing to mature to stage III can lead to the accumulation of PGs and their metabolites in the ovary (Clelland & Peng, 2009; Wu et al., 2000). In our study, we observed that cold temperature stimulation led to elevated levels of prostaglandins and androgens, including androstenedione and testosterone. Based on these observations, we hypothesize that the increased androgen levels may constitute a primary inhibitory factor affecting oocyte production and maturation.

Bile acids are synthesized from cholesterol in the liver and subsequently secreted into the intestinal tract to facilitate lipid absorption (Norlin & Wikvall, 2007). Despite their primary synthesis in the liver, bile acids are also linked to ovarian processes. Liu et al. (2003) demonstrated substantial expression of liver receptor homologue 1 (LRH-1), a key participant in cholesterol metabolism and bile acid synthesis, within rat ovaries. Cheng et al. (2021) discovered that late atretic follicles in the Chinese buffalo accumulate substantial amounts of bile acid-related substances in their follicular fluid. Smith et al. (2009) confirmed the existence of bile acid synthesis pathways within human ovaries. Our study revealed that low-temperature exposure significantly increased bile acid content in zebrafish ovaries. Similarly, Worthmann et al. (2017) found that cold exposure in mice accelerates the conversion of cholesterol to bile acids, accompanied by increased heat production.

Bile acids promote ovarian failure

It is important to recognize that bile acids possess systemic endocrine functions that modulate triglycerides, cholesterol, energy, and glucose homeostasis (Houten et al., 2006). In this study, administering GDCA to zebrafish resulted in ovarian failure, characterized by slower oocyte development and a reduced number of mature oocytes. This phenotype mirrors the effects observed in zebrafish ovaries under cold stress, suggesting that elevated bile acid levels may constitute a notable determinant in ovarian failure during cold stress. A similar association is found in human reproductive disorders, such as PCOS, characterized by chronic anovulation, hyperandrogenism, and polycystic ovarian morphology, where elevated bile acid levels within the follicular fluid are accompanied by increased serum concentrations of total testosterone and androstenedione (Yang et al., 2021; Zhang et al., 2019). This similarity to the metabolic perturbations observed in zebrafish ovaries exposed to low temperatures highlights the potential role of bile acids and unbalanced steroid hormones in ovarian failure.

In summary, we identified significant metabolite alterations in zebrafish ovaries under cold stress. Notably, changes in protein and lipid metabolism, reduced antioxidant levels, disrupted steroid hormones, and elevated bile acids collectively contributed to ovarian failure. Our findings provide new perspectives into the mechanisms of metabolic rebalance between survival and reproduction in ectothermic fish facing cold stress.

SUPPLEMENTARY DATA

Supplementary data to this article can be found online.

zr-45-791-S1.zip (3.7MB, zip)

Acknowledgments

COMPETING INTERESTS

The authors declare that they have no competing interests.

AUTHORS' CONTRIBUTIONS

W.H.L. and L.B.C. conceived and designed the research; Z.Q.L., M.D.B., and J.Z.L. performed the experiments; W.H.L., Z.Q.L., and M.D.B. analyzed the data; Z.Q.L., M.D.B., and L.B.C. wrote the manuscript. All authors read and approved the final version of the manuscript.

ACKNOWLEDGEMENTS

We thank Qiang-Hua Xu from Shanghai Ocean University for technical assistance during the study.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32130109) and Open Project Fund from Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science (OF2019NO01)

DATA AVAILABILITY

All Illumina RNA sequencing data from this project were deposited in the Genome Sequence Archive (GSA) database under accession number CRA015402, Science Data Bank (DOI: 10.57760/sciencedb.16619), and NCBI under BioProjectID PRJNA1020641. The metabolome data that support the findings of this study were deposited in the CNGB Sequence Archive (CNSA) of the China National GeneBank Database (CNGBdb) under accession number CNP0004915.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data to this article can be found online.

zr-45-791-S1.zip (3.7MB, zip)

Data Availability Statement

All Illumina RNA sequencing data from this project were deposited in the Genome Sequence Archive (GSA) database under accession number CRA015402, Science Data Bank (DOI: 10.57760/sciencedb.16619), and NCBI under BioProjectID PRJNA1020641. The metabolome data that support the findings of this study were deposited in the CNGB Sequence Archive (CNSA) of the China National GeneBank Database (CNGBdb) under accession number CNP0004915.

Articles from Zoological Research are provided here courtesy of Editorial Office of Zoological Research, Kunming Institute of Zoology, The Chinese Academy of Sciences

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