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. 2026 Feb 3;27:246. doi: 10.1186/s12864-026-12597-y

Transcriptomic and metabolomic analysis of metabolite changes and regulatory mechanisms associated with gonadal degeneration in yellow oil crab (Scylla paramamosain)

Biwei Wu 1,2, Jianye Chang 2, Hailin Liu 2,✉, Xiaolin Huang 3,4,5,✉, Jue Ruan 1,2,✉
PMCID: PMC12958495  PMID: 41634545

Abstract

Background

The yellow oil crab is a highly valuable aquatic species, with the accumulation of nutritional and flavor compounds closely linked to the degree of gonadal degeneration. However, the molecular mechanisms of gonadal degeneration remain unclear. In this study, we analyzed the differences in gene expression and metabolite accumulation across three gonadal degeneration stages (QX, GX, and TSX) in yellow oil crab using transcriptome and non-targeted metabolomics approaches, and identified key genes and metabolites involved.

Results

A total of 240 differential accumulated metabolites (DAMs) were identified, most of which were significantly more highly accumulated in GX and TSX than in QX. K-means clustering analysis of DAMs and gene expression data revealed distinct stage-specific expression patterns from QX to TSX stage. Moreover, the “steroid hormone biosynthesis” pathway was significantly enriched, with 15 highly expressed steroid hormones and their derivatives in GX and TSX. 7 types of key genes involved in steroid hormone biosynthesis (such as UGT, HSD17B12, STS, HSD3B, SULT1E1, SRD5A1, and CYP1A1) showed a trend of upregulation in GX and TSX stages. Gene-metabolite regulatory network analysis revealed significant correlations between 14 steroid hormones and 13 structural genes related to their biosynthesis (|r| > 0.8, p < 0.05).

Conclusions

Therefore, the identified differential steroid hormones and seven key genes were positively associated with gonadal degeneration in yellow oil crab. These results offer a theoretical basis for understanding the formation and aquaculture of the yellow oil crab.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-12597-y.

Keywords: Yellow oil crab, Gonadal degeneration, Steroid hormones, Untargeted metabolomics, Transcriptomics

Introduction

Yellow oil crab is a novel variant found exclusively in female mud crab (Scylla paramamosain), widely distributed along the coasts of East and Southeast Asia. Its hepatopancreas, muscles, and gonads not only have a pleasant aroma and delicious taste, but also possess high nutritional value [1, 2]. The oocytes of female mud crab (QX) cannot absorb vitellogenin, resulting in abnormal ovarian development and the excessive accumulation of lipids and lipophilic substances like carotenoids. Consequently, golden-yellow to reddish-orange oily substances appear in the crab meat and roe, hence the name “yellow oil crab” [3, 4]. The gonads of yellow oil crab are much smaller than those of QX, and their gonads undergo degeneration [5]. This degeneration leads to differences in the nutritional substance content of yellow oil crab compared to QX, which affects the formation and accumulation of flavor and nutrients [1]. Studies have shown that, compared to female mud crab, yellow oil crab has higher protein levels and aldehyde content in their edible tissues [4].

The degree of gonadal development in yellow oil crab directly influences their reproductive capacity and market value [6]. The yellow oil crab is a special physiological state resulting from abnormal gonadal development in QX. QX have plump gonads that range from orange-red to dark red, and gonads have not undergone degeneration. Yellow oil crab can be classified into two morphotypes based on the degree of gonadal degeneration and the “yellow oil” accumulation. In the Gaoyou yellow oil crab (GX), the gonads are partially degenerated, and “yellow oil” substances start to appear in the crab meat and roe. In the Toushou yellow oil crab (TSX), the gonads are mostly degenerated, and “yellow oil” spreads throughout almost the entire body, including the joints of the chelae and walking legs. Yellow oil crabs are known as the “top grade of crabs,” and their market prices vary significantly depending on morphology. The TSX is rich in oil and has a smooth, fragrant taste. Its price is two to three times higher than that of QX. The formation ratio is extremely low, and the formation mechanism remains unknown [1]. Therefore, a comprehensive analysis of differences in critical metabolites and regulatory genes during the different stages of gonadal degeneration (QX, GX, and TSX) is necessary to understand the physiological mechanisms behind the gonadal development of yellow oil crab.

The gonadal development of yellow oil crab is affected by various factors, including breeding conditions (such as salinity and temperature) and endocrine regulation (such as ecdysone and methyl farnesoate) [5, 7, 8]. Steroid hormones, such as estradiol and progesterone, play a central role in reproductive regulation by controlling the biosynthesis and metabolism of vitellogenin and promoting the growth and maturation of oocytes [9]. Studies have shown that in Eriocheir sinensis, 17β-estradiol is primarily found in the oocytes and follicle cells of the ovary, and its levels increase significantly with ovarian development and oocyte growth maturation [10]. In Penaeus monodon and Cherax albidus, estradiol levels increase during the peak of vitellogenesis and decrease after the gonads release mature oocytes [11, 12]. However, the regulatory mechanisms underlying the expression of steroid hormones during different stages of gonadal degeneration remain unclear, which limits the optimization of artificial breeding techniques. In addition, the biosynthesis of steroid hormones involves multiple enzymes. Recent studies have shown that in S. paramamosain, 3β-hydroxysteroid dehydrogenase (HSD3B) is a key enzyme, and the expression of its gene (SpHsd3b) is closely associated with gonadal development [13]. Studies involving RNA interference (RNAi) and inhibitors such as trilostane have shown that functional inhibition of HSD3B leads to decreased progesterone levels in the hemolymph [13]. The activity of HSD3B and other enzymes involved in steroid hormone biosynthesis, such as HSD17B and HSD20A, has also been found in the ovaries of Cancer Pagurus [14]. Therefore, the expression of steroid hormones and related genes may have important regulatory effects on gonadal development in yellow oil crab by affecting the metabolism and maturation of germ cells.

Although recent studies have gradually deepened our understanding of the role of steroid hormones in the gonadal development of crustaceans, some issues remain unresolved regarding their biosynthetic pathways, regulatory networks, and functional mechanisms compared to vertebrates. The molecular mechanism by which steroid hormones regulate the gonadal development of the yellow oil crab has not been systematically elucidated. The main goal of this study is to identify differences in gene expression and metabolite accumulation across three stages of gonadal degeneration (QX, GX, and TSX) through integrated transcriptomic and untargeted metabolomic analyses, and to elucidate the molecular mechanisms of steroid hormone biosynthesis and regulation. The findings aim to offer a theoretical foundation for understanding yellow oil crab formation and enhancing aquaculture management strategies.

Materials and methods

Collection of animal samples

The yellow oil crabs were collected from a crab farm in Nansha, Guangzhou, Guangdong Province, China. The rearing conditions included a water conductivity of 7.82 mS/cm, salinity of 0.48%, dissolved oxygen of 16.6 mg/L, and a pH of 8.0. A total of 15 healthy and intact crabs (approximately 200 g each) were collected. Each group of QX, GX, and TSX consists of 5 crabs. Place the crabs in an ice water bath for 20 min to make them lose consciousness, then carefully dissect on ice using forceps. Gonadal tissues are rapidly collected, immediately immersed in liquid nitrogen, and stored at − 80 °C for further analysis.

Metabolite extraction

Frozen samples are used for metabolomics analysis. The samples were taken out of the − 80 °C freezer and thawed on ice (all subsequent procedures were performed on ice). Each sample was thoroughly ground under liquid nitrogen, and 100 mg (± 5 mg) of gonadal tissue from each group—QX, GX, and TSX—was weighed into a 2 mL centrifuge tube. 1 mL of extraction solvent (methanol: water = 80:20, v/v) was added, and the mixture was vortexed at 1500 r/min for 5 min, then left to stand on ice for 15 min. The samples were then centrifuged at 12,000 r/min for 10 min at 4 °C. A volume of 500 µL of the supernatant was transferred to a new, correspondingly labeled centrifuge tube and left to stand at − 20 °C for 30 min, followed by a second centrifugation at 12,000 r/min for 3 min at 4 °C. Next, 200 µL of the supernatant was transferred to an autosampler vial with an inner liner for instrumental analysis. 10 µL aliquots from each sample were combined to prepare a pooled quality control (QC) sample.

Metabolomics analysis

UHPLC-MS/MS analysis was conducted using a quadrupole-electrostatic field orbitrap high-resolution liquid chromatography-mass spectrometry system (Thermo Scientific, Waltham, MA, USA). The Waters ACQUITY UPLC HSS T3 C18 chromatographic column (2.1 mm × 100 mm, 1.8 μm; Waters Corporation, Milford, Massachusetts, USA) was used. The column temperature was maintained at 40 °C, and the flow rate was set at 0.3 mL/min. The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The injection volume was 4 µL. The gradient elution program was as follows: 0–2 min, 5%–20% solvent B; 2–5 min, 20%–60% B; 5–7.5 min, 60%–99% B; 7.6–10 min, 5% B. Both positive and negative ion modes were acquired within a range of 80–1200 m/z. The sheath gas flow rate was set at 35 arb, and the auxiliary gas flow rate at 15 arb. The auxiliary gas heater temperature was set to 310 °C, the capillary temperature to 320 °C, and the spray voltage for both positive and negative modes was 3.20 kV.

Identified metabolites were annotated using the KEGG database (https://www.genome.jp/kegg/), the HMDB database (https://hmdb.ca/metabolites), and the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Difference analysis for metabolites was performed using R software (v 4.4.2, https://www.R-project.org/). Differentially accumulated metabolites (DAMs) were identified based on fold change (FC) and unpaired two-tailed Student’s t-tests (|log2FC| ≥ 2, p < 0.05) [15]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R software package MetaboAnalystR v1.0.1 [16].

Transcriptome sequencing and analysis

RNA extraction, sequencing, and transcriptome analysis were performed on five biological replicates from gonadal tissues of QX, GX and TSX. According to the manufacturer’s instructions, total RNA from each group was extracted using Trizol reagent (Invitrogen, USA). RNA quantification was performed as described in reference [3]. High-quality RNA samples from each group were sent to BGI (Shenzhen, China) for library construction and sequencing on the DNBSEQ-T7 platform, producing 150 bp paired-end reads. Each group consisted of five biological replicates.

Raw sequencing data were filtered using FastQC (v0.19.3, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic (v0.32) [17] to remove adapter sequences and low-quality reads. Clean reads were then aligned to the S. paramamosain reference genome using HISAT2 (v2.2.4) [18]. Fragments per kilobase per million total mapped fragments (FPKM) was used to measure gene expression levels, and differential transcript expression analysis was performed using the DESeq2 package (v1.24.0) [19]. Screening conditions for differentially expressed genes (DEGs) were |log2 fold change| ≥ 1 and false discovery rate < 0.05. The ClusterProfiler (v4.6.0) [20] R software package was used to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on DEGs.

Multivariate statistical analysis

Gene and metabolite expression values were normalized using the Z-score normalization method. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed using R software (www.r-project.org/ ) with default settings. K-means clustering analysis was conducted using R software’s function package stats (v 4.2.0, https://search.r-project.org/R/refmans/stats/html/00Index.html). Pearson correlation analysis was used to construct the regulatory network between genes and metabolites [21]. Correlations between genes and metabolites were calculated using R software, and all associations were visualized using Cytoscape software [22].

Quantitative real-time PCR analysis

In this study, 24 differentially expressed genes (DEGs) involved in the steroid biosynthesis pathway were selected for validation by RT-qPCR. RT-qPCR experiments were performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a CFX Opus 96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). β-actin is used as an internal control for the normalization of gene expression [3]. The primers were designed using Primer-Blast of NCBI (https://www.ncbi.nlm.nih.gov ), and they are listed in Table S1. To determine the relative fold changes in mRNA expression levels, gene expression was calculated using the 2−ΔΔCt method. Each sample included three biological replicates, and six technical replicates were performed for each biological replicate. According to Tukey’s multiple comparison test, different letters indicate significant differences between treatments.

Results

Metabolomic analysis of gonadal degeneration stages in yellow oil crabs

We performed non-targeted metabolomic analysis on the three samples groups (Fig. 1A). During the three stages of gonadal degeneration, a total of 703 compounds were identified, including 165 lipids and lipid-like molecules, 134 organoheterocyclic compounds, 127 organic acids and derivatives, 117 benzenoids, 44 phenylpropanoids and polyketides, 46 organic oxygen compounds, 26 organic nitrogen compounds, 22 nucleosides, nucleotides, and analogs, 9 alkaloids and derivatives, 5 organosulfur compounds, and 8 metabolites belonging to other groups categories (Fig. 1B, Table S2). Additionally, principal component analysis (PCA) was conducted, showing that all biological replicates clustered together and that the 703 identified metabolites could be grouped into three categories, indicating high reliability of the generated metabolomic data (Fig. 1C).

Fig. 1.

Fig. 1

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Metabolomics analysis of the three stages of gonadal degeneration. A Three stages of gonadal degeneration. B Classification of 703 metabolites. C PCA analysis of metabolomics data. D Venn diagrams comparing paired DAMs. E Clustering heat map of 240 DAMs. F KEGG pathway analysis of QX vs. GX. G KEGG pathway analysis of GX vs. TSX. H KEGG pathway analysis of QX vs. TSX. Scale bar = 10 mm. The pathways highlighted in red letters were significantly enriched in all three pairwise comparisons (QX vs. GX, GX vs. TSX, and QX vs. TSX)

The 703 compounds were annotated according to HMDB, PubChem, KEGG, and other databases, and a difference analysis was performed. As a result, 240 differential metabolites (DAMs) were identified across the three groups (Table S3). There were 165, 106, and 173 DAMs in the QX vs. GX, QX vs. TSX, and GX vs. TSX comparison groups, respectively, with a total of 30 DAMs across all three groups (Fig. 1D). Hierarchical clustering analysis (HCA) was conducted to show the relative abundance of DAMs and the relationships among different sample groups. As shown in Fig. 1E, three main clusters were identified based on differences in the relative contents of metabolites. This suggests that distinct metabolic accumulation patterns exist across the three stages of gonadal degeneration in yellow oil crab. The metabolites in cluster A showed an upregulation trend in GX, those in cluster B were upregulated in TSX, and those in cluster C were upregulated in both QX and TSX. In TSX, the levels of lipids and lipid-like molecules were significantly increased, which might indicate a correlation between lipid accumulation and the degree of gonadal degeneration. In addition, KEGG enrichment analysis showed that DAMs were linked to multiple metabolic pathways. The results showed that “arginine and proline metabolism”, “β − Alanine metabolism”, and “histidine metabolism” were significantly enriched in all three comparison groups. The “steroid hormone biosynthesis” pathway was significantly enriched in QX vs. GX and GX vs. TSX groups (Fig. 1F-H).

Gene expression analysis of the gonadal degeneration stage in yellow oil crabs

To investigate the transcriptional regulation of genes during the three stages of gonadal degeneration in yellow oil crab, transcriptome sequencing was performed on three sample groups: QX, GX, and TSX. Principal component analysis (PCA) based on fragments per kilobase of exon per million mapped reads (FPKM) values showed that all biological replicates clustered together, and there was a significant separation among the three groups of samples, indicating that our sequencing data is highly reliable (Fig. 2A). 27,158 genes were identified in three stages of gonadal degeneration (Table S4). In the QX vs. GX comparison, 3,826 DEGs were found, including 2,325 upregulated and 1,501 downregulated genes. The QX vs. TSX comparison revealed 6,284 DEGs, with 4,409 upregulated and 1,875 downregulated. Between GX and TSX, 5,740 DEGs were identified, consisting of 3,599 upregulated and 2,141 downregulated genes (Fig. 2B, Table S5-7). The largest number of differentially expressed genes (DEGs) was identified in QX vs. TSX comparison. A total of 657 DEGs were identified across the three comparison groups, suggesting that these core conserved genes may be involved in gonadal degeneration in yellow oil crab (Fig. 2C).

Fig. 2.

Fig. 2

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Gene expression analysis across three stages of gonadal degeneration. A PCA of transcriptome data. B The number of differentially expressed genes (DEGs). C DEG pairwise comparison Venn diagrams. D GO terms. E KEGG pathway analysis of QX vs. GX. F KEGG pathway analysis of GX vs. TSX. G KEGG pathway analysis of QX vs. TSX. The pathways highlighted in red letters were significantly enriched in all three pairwise comparisons (QX vs. GX, GX vs. TSX, and QX vs. TSX)

Additionally, Based on GO functional analysis, the DEGs were significantly enriched in categories related to molecular function, cellular component, and biological process (Fig. 2D). KEGG enrichment analysis was performed to explore the potential functions of DEGs. The results showed that, DEGs were significantly enriched in multiple pathways related to signal transduction, hormone biosynthesis, and material metabolism. Among the top 20 KEGG pathways, “Neuroactive ligand–receptor interaction”, “Steroid hormone biosynthesis”, “Ovarian steroidogenesis”, “Hippo signaling pathway”, “Apoptosis”, and “Carbohydrate digestion and absorption” were common pathways identified in the QX vs. GX, GX vs. TSX, and QX vs. TSX (Fig. 2E-G). These common pathways are mainly associated with neuroendocrine signal transduction, steroid hormone metabolism, cell proliferation, and programmed cell death. The “neuroactive ligand–receptor interaction” pathway contained 42, 45, and 56 DEGs in the QX vs. GX, GX vs. TSX, and QX vs. TSX comparisons, respectively. The “steroid hormone biosynthesis” pathway was enriched with 19, 23, and 18 DEGs, while the “Hippo signaling” pathway included 21, 30, and 42 DEGs. The “apoptosis” pathway contained 21, 31, and 45 DEGs, and “ovarian steroidogenesis” was enriched with 11, 15, and 18 DEGs in the respective comparisons. In the QX vs. GX, DEGs enrichment involves neural-receptor signaling, steroid hormone biosynthesis, carbohydrate metabolism, and regulation of cellular signaling pathways. In the GX vs. TSX, in addition to hormone-related pathways, it also involved pathways such as the cytoskeleton, energy metabolism, and pancreatic secretion. Compared with the other two comparisons, the QX vs. TSX comparison additionally showed significant enrichment of the MAPK, cAMP, Ras, GnRH, and thyroid hormone signaling pathways. This indicates that the differentially expressed genes that appear at different stages of gonadal degeneration are involved in a variety of biological processes related to gonadal function and structural remodeling.

Clustering and correlation analysis of DAM and DEG in the three stages of gonadal degeneration

To further explore the relationship between the expression patterns of differentially expressed genes (DEGs) and the accumulation of differentially metabolites (DAMs) across the three stages of gonadal degeneration, the 240 DAMs were grouped into four clusters using the K-means clustering algorithm (Fig. 3A, Table S8). The results showed that metabolites in Clusters 2 and 3 were mainly accumulated in QX, while 86 metabolites in Cluster 4 were predominantly accumulated in GX. Notably, 84 metabolites in Cluster 1 were highly accumulated in TSX, including lipids and lipid-like molecules (19 species), organoheterocyclic compounds (17 species), organic acids and derivatives (16 species), benzenoids (15 species), phenylpropanoids and polyketides (6 species), nucleosides, nucleotides, and analogues (4 species), organic nitrogen compounds (3 species), and organic oxygen compounds (4 species)(Table S8). In contrast, the 23 metabolites in Cluster 2 showed a decreasing trend with gonadal degeneration, reaching the lowest levels at the TSX stage. These included organoheterocyclic compounds (7 species), organic acids and derivatives (5 species), nucleosides, nucleotides, and analogs (3 species), organic oxygen compounds (3 species), lipids and lipid-like molecules (2 species), Phenylpropanoids and polyketides (2 species), and organic nitrogen compounds (1 species)(Table S8).

Fig. 3.

Fig. 3

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Trend analysis of DAM and DEG clustering and Pearson correlation analysis. A Clustering trend analysis of 204 DAMs. B Clustering trend analysis of DEGs. C A heat map showing the correlation between the gene module and the data of the 84 DAMs

We used the same K-means method to perform cluster analysis on the FPKM data from QX, GX, and TSX, and identified four clusters of differentially expressed genes (Fig. 3B, Table S9). The results showed that these genes exhibited significantly different expression patterns from QX to TSX, with genes in Clusters 2, 3, and 4 being downregulated at the TSX stage. However, the 5,223 genes in Cluster 1 showed increased expression levels from the QX to TSX stage, indicating a positive correlation with the degree of gonadal degeneration in yellow oil crab. On the contrary, 4,758 genes in cluster 2 were gradually downregulated, and they were negatively correlated with the degree of gonadal degeneration. The genes in Clusters 3 and 4 show high expression in GX, which may imply that these genes have a positive correlation with the early stage of gonadal degeneration in yellow oil crab. Among these, the 24 key genes involved in steroid hormone metabolism were distributed across four expression clusters (Table S10). Specifically, multiple members of the UGT (LOC135109029, LOC135089202, LOC135110610, and LOC135109064), SRD5A1 (LOC135112076), HSD17B12 (LOC135097787, LOC135099700, LOC135099702, and LOC135106883), and SULT1E1 (LOC135097616, LOC135110077, LOC135100246, LOC135116141, LOC135116438, LOC135110106, and LOC135109474) were primarily clustered into Clusters 3 and 4, which were characterized by relatively high expression levels at the gonadal development stage (GX), followed by marked downregulation at the gonadal degeneration stage (TSX). In contrast, genes in Cluster 1 was upregulated in GX and TSX stage, mainly including UGT (LOC135089205, LOC135089171, and LOC135089201), HSD3B (LOC135105155 and LOC135091793), STS (LOC135109873), and CYP1A1 (LOC135115794).

To further verify the correlation between genes and metabolites, we calculated the Pearson correlation coefficients between the average expression value of each cluster of genes in three groups of samples (QX, GX, TSX) and 84 metabolites. The 5,223 genes in Cluster 1 were significantly positively correlated with the 84 metabolites highly accumulated in the TSX stage, suggesting a coordinated pattern of gene expression and metabolite accumulation during gonadal degeneration. (Fig. 3C, Table S11).

Identification of steroid hormones and genes related to steroid hormone biosynthesis

In this study, fifteen differentially accumulated steroids and their derivatives were identified at three stages of gonadal degeneration (Fig. 4B, Table S12). Heat map analysis shows that metabolites exhibit a clear stage-specific accumulation pattern. Specifically, the expression of eight steroid hormones and derivatives is upregulated in the gonads of the GX, including urocortisol, estrone 3-sulfate, 17a-Hydroxyprogesterone, 11-Deoxycortisol, pregnenolone sulfate, corticosterone, testosterone, and estrone. Seven of these were upregulated in the gonads of the TSX, including 11-Deoxycorticosterone, estrone glucuronide, cortisone, estriol, Progesterone, testosterone glucuronide, and pregnenolone (Fig. 4A and B).

Fig. 4.

Fig. 4

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Schematic diagram of the steroid hormone biosynthesis pathway across three stages of gonadal degeneration. A Fifteen differentially accumulated steroid hormones and their derivatives identified are highlighted in red, while the differentially expressed genes (LOC IDs) identified are presented in black italics, and the corresponding enzyme abbreviations are shown in green. Solid arrows indicate single-step catalytic reactions, whereas dashed arrows represent multi-step reactions. Metabolite names and enzyme abbreviations shown in black alongside the arrows indicate metabolites or genes that were either not detected or detected but did not exhibit significant differences. B Heat map showing the relative abundance of the 15 differentially accumulated steroid hormones and their derivatives across the three stages of gonadal degeneration. UGT, UDP-glucuronosyltransferase; SULT1E1, estrone sulfotransferase 1; SD17B12, 17β-hydroxysteroid dehydrogenase type 12; STS, steroid sulfatase; HSD3B, 3β-hydroxy-Δ5-steroid dehydrogenase; CYP1A1, cytochrome P450 1A1; SRD5A1, steroid 5α-reductase type 1; CYP21A, steroid 21-hydroxylase

KEGG pathway analysis based on DAMs and DEGs revealed that the “steroid hormone biosynthesis” pathway was significantly enriched in all three stages of gonadal degeneration (Figs. 1F–H and 2E–G). 24 annotated genes related to “steroid hormone biosynthesis” pathway were identified, belonging to seven enzyme families. These included 7 UDP-glucuronosyltransferases (UGT), 7 estrone sulfotransferases (SULT1E1), 4 17β-hydroxysteroid dehydrogenase type 12s (HSD17B12), 2 steroid sulfatases (STS), 2 3β-hydroxy-Δ5-steroid dehydrogenases (HSD3B), 1 cytochrome P450 1A1 (CYP1A1), and 1 steroid 5α-reductase type 1 (SRD5A1) (Fig. 4A, Table S13).

Seven UGT genes, including LOC135109029, LOC135089202, LOC135110610, LOC135109064, LOC135089205, LOC135089171, and LOC135089201, showed significant differential expression across different stages. Among them, LOC135089205, LOC135089171, and LOC135089201 exhibited gradually increased expression in GX and TSX, consistent with the rising levels of testosterone glucuronide and estrone glucuronide in GX and TSX (Fig. 4A and B). These genes encode members of the UDP-glucuronosyltransferase (UGT) family, which are known to catalyze the conjugation of glucuronic acid to steroid hormones, forming their glucuronide derivatives. This process facilitates hormone inactivation and clearance, potentially contributing to the reduced levels of free testosterone and estrone. CYP1A1 (LOC135115794), is a key enzyme in estradiol metabolism that decreases free estradiol levels through hydroxylation, producing the less active form estriol. The two HSD3B (LOC135105155, LOC135091793) genes were significantly upregulated during the TSX stage, consistent with the increase in progesterone content. The seven SULT1E1 genes were significantly upregulated in GX, consistent with the high accumulation of estrone 3-sulfate at this stage (Fig. 4A and B). SULT1E1 sulfates Estrone into the biologically inactive Estrone 3-sulfate to lower estrogen levels. In GX and TSX, the expression of STS increased, along with a gradual rise in pregnenolone levels.

Additionally, the genes SRD5A1, HSD17B12, and SULT1E1 were mainly upregulated in the GX but downregulated in the TSX. This expression pattern suggests that the enzymatic activities encoded by these genes are higher during the early stage of gonadal degeneration and decrease in the later stage (Fig. 4A).

Steroid hormone biosynthesis regulation network

A Pearson correlation analysis was performed between the 15 identified steroid hormones and derivatives and 24 structural genes. The results showed that 15 steroid hormones and derivatives were significantly positive correlation with multiple structural genes (P < 0.05) (Fig. 5A). Correlation network analysis identified significant positive or negative correlations between 14 steroid hormones and 13 structural genes, totaling 73 correlated pairs (51 positive and 22 negative) (|r| > 0.8, P < 0.05) (Fig. 5B, Table S14). Progesterone showed a very strong correlation (r = 0.976) with an STS(LOC135113565). This gene also had significant positive correlations with nine other steroid hormones and derivatives, including urocortisol (r = 0.970), 11-Deoxycortisol (r = 0.963), 17α-Hydroxyprogesterone (r = 0.963), pregnenolone sulfate (r = 0.961), estrone (r = 0.950), estrone 3-sulfate (r = 0.947), pregnenolone (r = 0.934), testosterone (r = 0.874), and testosterone glucuronide (r = 0.870). These findings suggest that STS(LOC135113565) may play a central role in steroid hormone biosynthesis or metabolic regulation. Estrone, progesterone, pregnenolone, pregnenolone sulfate, and urocortisol showed significant positive correlations (r > 0.8, p < 0.05) with not only STS (LOC135113565), but also three SULT1E1 (LOC135110106, LOC135116141, LOC135100246), and two HSD17B12 (LOC135097787, LOC135106883). These findings suggest that these genes may work together in the biosynthesis, conversion, and inactivation of steroid hormones, and play a significant role in regulating steroid hormones.

Fig. 5.

Fig. 5

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Correlation between the expression levels of key genes in steroid biosynthesis and steroid content, as well as the regulatory network of key genes. A The correlation between the expression levels of key genes and steroid content. B The regulatory network of essential genes involved in “steroid hormone biosynthesis” pathway. The green and orange graphics represent structural genes and metabolites respectively. The red and gray lines respectively represent positive (r > 0.8) and negative (r < -0.8) correlations. The gene names corresponding to the LOC ID shown in the figure can be found in Table S14 of the supplementary materials

Validation of transcriptomic data

RT-qPCR experiments were conducted on 24 candidate genes involved in “steroid hormone biosynthesis” pathway, including 7 UGTs, 7 SULT1E1s, 4 HSD17B12s, 2 STSs, 1 CYP1A1, 1 SRD5A1, and 2 HSD3Bs. Data analysis showed that four UGTs (LOC135109029, LOC135089202, LOC135110610, LOC135109064), one SRD5A1 (LOC135112076), one STS (LOC135113565), four HSD17B12, and seven SULT1E1 were highly expressed in GX, consistent with the upregulation trend observed in the gene expression heatmap from the RNA-Seq data (Figs. 4A and 6A, B, D, F and G). Additionally, one CYP1A1 (LOC135115794), one STS (LOC135109873), three UGT (LOC135089205, LOC135089171, LOC135089201), and two HSD3B genes showed high expression in the TSX group, which aligned with the upregulation trend observed in the TSX samples in the RNA-Seq heatmap (Figs. 4A and 6A, B, C and E).

Fig. 6.

Fig. 6

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Analysis of the relative expression of genes related to steroid hormone biosynthesis by RT-qPCR. A The relative expression levels of the seven UGT genes. B The relative expression levels of two STS genes. C The relative expression level of one CYP1A1 gene. D The relative expression levels of seven SULT1E1 genes. E The relative expression levels of two HSD3B genes. F The relative expression level of one SRD5A1 gene. G The relative expression levels of four HSD17B12 genes. Error bars indicate the standard deviation of three biological replicates. Different letters indicate significant differences between different treatments according to Tukey’s multiple comparisons test

Discussion

Yellow oil crab is a highly nutritious seafood primarily eaten in East Asia, especially in China, Singapore, and Vietnam. It is rich in protein, lipids, various vitamins (such as vitamins D, E, and B complex), and trace elements like calcium, iron, and zinc [4]. These nutritional components not only provide abundant nutrients for the human body but also help boost physical immunity. Gonadal degeneration is a vital physiological process in the life cycle of the S. paramamosain, directly affecting its growth, reproductive ability, and commercial worth. In this study, combined transcriptomic and untargeted metabolomic analyses were conducted on the three stages of gonadal degeneration during yellow oil crab formation to systematically reveal the regulatory network of steroid hormone biosynthesis during this process. This work provides new theoretical insights into the molecular mechanisms underlying reproductive development in crustaceans.

We used untargeted metabolomics based on high-resolution UHPLC-MS/MS to analyze the metabolite differences in the three stages of gonadal degeneration (QX, GX, and TSX) during yellow oil crab formation. Previous studies have identified elevated levels of unsaturated fatty acids in yellow oil crab muscle and higher contents of essential amino acids and aldehydes in the hepatopancreas [3, 4]. However, investigations into metabolite accumulation during gonadal degeneration in yellow oil crab remain limited. In this study, we identified a total of 703 metabolites, with 240 showing significant differences across the three stages of gonadal degeneration (QX, GX, and TSX). These differential metabolites were subjected to K-means analysis, and the results revealed the stage-specifics at the metabolic level during each stage. The stage-specific accumulation of these DAMs covers multiple functional categories suggests that gonadal degeneration in the yellow oil crab involves complex metabolic network regulation and could be influenced by other physiological or environmental factors(Fig. 3A, Table S8). Compared with previous reports [4, 23, 24], we found that metabolite accumulation was higher in the GX and TSX stages (Fig. 1E). KEGG enrichment analysis of differentially accumulated metabolites (DAMs) revealed that the “steroid hormone biosynthesis” pathway was significantly enriched in both QX vs. GX and GX vs. TSX comparisons suggesting that this pathway is positively related to the biosynthesis and metabolism changes of hormones during the process of gonadal degeneration. Enrichment of various amino acid biosynthesis and metabolism pathways indicates that proteins and amino acids are essential during gonadal development (Fig. 1F-H).

Additionally, through transcriptome analysis, we identified differences in gene expression during gonadal degeneration. KEGG enrichment analysis of differentially expressed genes (DEGs) from the QX vs. GX, GX vs. TSX, and QX vs. TSX comparisons revealed six metabolic pathways common to all three groups (Fig. 2E-G). Among these, the “neuroactive ligand-receptor interaction” pathway may influence reproductive-related neuroendocrine signaling. This is consistent with previous findings that crustacean neuropeptides regulate reproductive behavior and gonadal differentiation by activating G protein-coupled receptors [25, 26]. The important role of the “Hippo signaling” pathway in crustacean immunity and regeneration has been well documented [27, 28]. During gonadal degeneration, “hippo signaling” pathway may play a key role in regulating oocyte proliferation and maintaining the balance between the homeostasis and apoptosis of gonadal cells. Previous studies have shown that apoptosis is an evolutionarily conserved physiological process regulated by various hormones and growth factors, involved in tissue remodeling, differentiation, and the degeneration of multiple cell types [29]. “Apoptosis” pathway probably controls the programmed death of gonadal cells during gonadal degeneration, helping with the cyclical clearance of oocytes and maintaining cellular balance. DEGs in the QX vs. TSX comparison were enriched in various signaling pathways. Compared to the signaling pathways enriched during ovarian development in Eriocheir sinensis, we also identified thyroid hormone, GnRH, and Ras signaling pathways [30]. This indicates that the coordinated regulation of multiple signaling pathways is associated with the process of gonad degeneration in the yellow oil crab. Notably, the enrichment of the “GnRH signaling” pathway may indicate that crustacean gonads possess a gonadotropin-releasing hormone regulatory axis similar to that of vertebrates, which could promote steroid hormone biosynthesis by activating the PKC-MAPK cascade [31].

Steroid hormones are the primary regulators of ovarian development and reproductive behavior in crustaceans [7]. In freshwater rice field crabs and red swamp crayfish, progesterone and 17β-estradiol promote ovarian maturation by stimulating the production and release of vitellogenin (Vg) [32–34]. However, 17β-estradiol was not detected in the untargeted metabolomic analysis of this study. This result may be attributed to the extremely low level of estradiol-17β in the crustaceans, and its concentration is likely to be lower than the detection limit of non-targeted metabolomics methods. Existing studies indicate that using LC-MS/MS, 17β-estradiol was positively detected in the brain tissues of Callinectes sapidus, however, the measured amount was below the method quantitation limit (150 pg/tissue, n = 3), suggesting that free 17β-estradiol may occur at extremely low levels [35]. Moreover, within the steroid hormone biosynthesis pathway, testosterone showed significant accumulation at the GX stage; however, the key enzyme catalyzing its conversion to 17β-estradiol, aromatase (CYP19), was not detected in our transcriptomic dataset. This observation is consistent with previous studies showing that CYP19 is a product of chordate evolution and has not been detected among the protostome invertebrates [36, 37]. This implies that crustaceans may lack the classical CYP19-mediated aromatization pathway that converts testosterone to estradiol. Therefore, even with substantial accumulation of precursors, 17β-estradiol cannot be stably synthesized via this pathway. This results in extremely low endogenous levels of 17β-estradiol. In addition, even if trace amounts of 17β-estradiol are present in crustaceans, existing studies have shown that Estradiol is quickly converted to estrone that can be metabolized in one of two ways. Estrone can be hydroxylated in one direction to 16–alpha hydroxyestrone or estriol [38, 39].

Research on the changes in steroid hormone metabolism and related gene expression during the gonadal degeneration stage is remain limited. This study identifyed 15 species of steroid hormones and derivatives in the process of gonadal degeneration (Fig. 4B, Table S11). Among these, testosterone, pregnenolone, progesterone, and estradiol have also been reported in studies on Nephrops norvegicus and Pandalus kessleri [40, 41]. In studies on ovarian development of Eriocheir sinensis, four genes involved in the biosynthesis of steroid hormones have been identified [30]. This study identified 24 annotated genes related to steroid hormone biosynthesis. These genes involve multiple regulatory steps, including steroid hormone conversion, glycosylation, and sulfation. Specifically, they include 7 UGT, 7 SULT1E1, 4 HSD17B12, 1 CYP1A1, 1 SRD5A1, 2 STS, and 2 HSD3B (Fig. 4A, Table S12). Among them, UDP-glucuronosyltransferases (UGT) are an important group of metabolic enzymes that play critical roles in diverse physiological processes such as detoxification, metabolic homeostasis, and physiological regulation [42]. In zebrafish studies, the UGT gene family has been divided into multiple subfamilies, and alternative splicing can produce different transcripts [43, 44]. During gonadal degeneration in the yellow oil crab, UGT enzymes may help deal with environmental stressors such as high temperatures and pollutants. This detoxification process is essential for the survival and development of these crabs in their natural habitat. In mouse studies, the SULT1E1 gene has been shown to inhibit and inactivate estrogen through sulfation [45]. SULT1E1 in the yellow oil crab may also inhibit the changes in estrogen activity through sulfation, and may be were positively correlated with the gonadal degeneration of the yellow oil crab.

The hydroxysteroid dehydrogenase (HSD) gene family regulates the biosynthesis and metabolism of steroids by catalyzing the reduction of 17-ketosteroids or the oxidation of 17β-hydroxysteroids [46, 47]. In the study of Nile tilapia and sturgeon, HSD17B12 was responsible for converting 17α, 17α-hydroxyprogesterone (17OHP) into the maturation steroid 17α, 20β-dihydroxy-4-progetene-3-one (DHP), which is essential for oocyte maturation [48, 49]. In this study, the four HSD17B12 genes were upregulated during the gonadal degeneration process (GX stages) and were positively associated with gonadal degeneration by regulating the conversion of 17β-estradiol (the active form) to estrone (the less active form). The HSD3B gene is crucial for producing sex steroid hormones such as progesterone, androgen, and estrogen [50]. Zebrafish studies have shown that knocking out HSD3B greatly reduces progesterone biosynthesis, causing an imbalance in the steroid metabolic pathway [51]. In this study, the expression of two HSD3B genes was upregulated during the gonadal degeneration process (TSX stages), which might have promoted progesterone biosynthesis. This expression pattern indicates that HSD3B was positively related with the metabolic changes of steroid hormones during the process of gonadal degeneration.(Fig. 4A). Steroid sulfatase (STS) catalyzes the hydrolysis of estrone sulfate and estradiol sulfate through desulfurization in zebrafish, thereby regulating the activity of steroid hormones [52]. In this study, STS expression was upregulated during the gonadal degeneration process (GX and TSX stages), which may promote the accumulation of free pregnenolone by catalyzing the hydrolysis of pregnenolone sulfate, thus supporting downstream progesterone biosynthesis, and was positively associated with gonadal degeneration in yellow oil crab (Fig. 4A). Although this study systematically revealed the regulatory network of steroid hormone biosynthesis during gonadal degeneration of the yellow oil crab, and identified key genes and metabolites, our work still has some limitations. This study mainly focuses on the association analysis of omics data and pathway enrichment. Although it predicted the potential functions of key genes (such as UGT, SULT1E1, HSD17B12, etc.) in steroid hormone metabolism, further molecular biology experiments (such as gene knockout, overexpression or enzyme activity determination) are needed to confirm the specific mechanism of these genes in the gonadal degeneration. Furthermore, the gonadal degeneration is a complex physiological process that may also involve other unexplored regulatory mechanisms, such as non-coding RNA regulation, epigenetic modifications, and the interaction of the neuroendocrine-immune network. Future research should combine functional experiments and multi-dimensional omics data to further elucidate the precise molecular regulatory mechanisms of gonadal degeneration, and provide a more comprehensive theoretical basis for the reproductive development biology of crustaceans.

Conclusion

This study conducted transcriptomic and untargeted metabolomic analyses across different stages of gonadal degeneration in the yellow oil crab. The results revealed distinct expression profiles of genes and metabolites, emphasizing the key role of steroid hormone biosynthesis in regulating gonadal degeneration. 240 differentially accumulated metabolites (DAMs) were identified in QX, GX, and TSX stages, with lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and derivatives being the main categories. Most DAMs showed higher expression in GX and TSX. Additionally, 27,158 differentially expressed genes (DEGs) were found, and clustering analysis demonstrated unique expression patterns across the three stages. In KEGG pathway analysis, “steroid hormone biosynthesis” pathway was significantly enriched among both DAMs and DEGs. 15 species of steroids and their derivatives were upregulated in GX and TSX stages, while key genes such as UGT, HSD17B12, STS, HSD3B, SULT1E1, SRD5A1, and CYP1A1 played vital regulatory roles in this pathway. This study provides new insights into the molecular mechanisms behind gonadal development and degeneration in the yellow oil crab, offering a theoretical basis for high-quality yellow oil crab aquaculture.

Supplementary Information

Supplementary Material 1. (8MB, xlsx)

Acknowledgements

Not applicable.

Abbreviations

UGT

UDP-glucuronosyltransferase

SULT1E1

Estrone sulfotransferase 1

HSD17B12

17β-hydroxysteroid dehydrogenase type 12

STS

Steroid sulfatase

HSD3B

3β-hydroxy-Δ5-steroid dehydrogenase

CYP1A1

Cytochrome P450 1A1

SRD5A1

Steroid 5α-reductase type 1

KEGG

Kyoto Encyclopedia of Genes and Genomes

GO

Gene Ontology

DEMs

Differentially expressed metabolites

DEGs

Differentially expressed genes

Authors’ contributions

WBW conceived the study, performed data analysis, developed methodology, conducted investigations, validated results, created visualizations, implemented software, and wrote the original draft. CJY contributed to methodology development and investigation. LHL acquired funding and participated in manuscript review and editing. HXL provided resources and contributed to manuscript review and editing. RJ provided resources, conceptualized the research framework, acquired funding, administered the project, supervised the work, and participated in manuscript review and editing. All authors read and approved the final manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (No.2022YFC3400300), the Natural Science Foundation of China (NO.32300518), and the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ZDRW202503)and (Grant No. CAAS-ZDRW202601).

Data availability

The RNA-Seq raw data have been deposited in the Genome Sequence Archive of the National Genomics Data Center (NGDC) and can be accessed under the accession number CRA028097 (https://ngdc.cncb.ac.cn/gsa/browse/CRA028097).

Declarations

Ethics approval and consent to participate

Our animal experiment research was conducted in accordance with the ARRIVE guidelines. All experimental animal procedures were reviewed and approved by the guidelines of Institutional Animal Care and Use Committee of Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, the animal Ethics Committee approval number is AGIS2020.04.17.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Hailin Liu, Email: liuhailin@caas.cn.

Xiaolin Huang, Email: huangxl@scsfri.ac.cn.

Jue Ruan, Email: ruanjue@caas.cn.

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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 Material 1. (8MB, xlsx)

Data Availability Statement

The RNA-Seq raw data have been deposited in the Genome Sequence Archive of the National Genomics Data Center (NGDC) and can be accessed under the accession number CRA028097 (https://ngdc.cncb.ac.cn/gsa/browse/CRA028097).

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