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Sci Rep
2021 Jan 26;111:2194. doi: 10.1038/s41598-021-81917-x.
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Sex steroids and steroidogenesis-related genes in the sea cucumber, Holothuria scabra and their potential role in gonad maturation.
Thongbuakaew T
,
Suwansa-Ard S
,
Chaiyamoon A
,
Cummins SF
,
Sobhon P
.
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The sea cucumber Holothuria scabra is an economically valuable marine species which is distributed throughout the Asia-Pacific region. With the natural population declining due to over fishing, aquaculture of this species is deemed necessary. Hence, it is essential to understand the mechanisms regulating the reproduction in order to increase their populations. Sex steroids, including estrogens, androgens and progestogens, play an important role in reproduction in most vertebrates and several invertebrates. It has been proposed that sea cucumbers have the same sex steroids as vertebrates but the steroidogenic pathway in the sea cucumbers is still unclear. In this study, we demonstrated by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that sex steroids (estradiol, progesterone, and testosterone) were present in H. scabra neural and gonadal tissues. In silico searches of available sea cucumber transcriptome data identified 26 steroidogenesis-related genes. Comparative analysis of encoded proteins for the steroidogenic acute regulatory protein (HscStAR), CYP P450 10, 17 and 3A (HscCYP10, HscCYP17, HscCYP3A) and hydroxysteroid dehydrogenases (Hsc3β-HSD, Hsc17β-HSD) with other species was performed to confirm their evolutionary conservation. Gene expression analyses revealed widespread tissue expression. Real-time PCR analysis revealed that HscStAR, HscCYP10, Hsc3β-HSD, and Hsc17β-HSD gene expressions were similar to those in ovaries and testes, which increased during the gonad maturation. HscCYP17 mRNA was increased during ovarian development and its expression declined at late stages in females but continued high level in males. The expression of the HscCYP3A was high at the early stages of ovarian development, but not at other later stages in ovaries, however it remained low in testes. Moreover, a role for steroids in reproduction was confirmed following the effect of sex steroids on vitellogenin (Vtg) expression in ovary explant culture, showing upregulation of Vtg level. Collectively, this study has confirmed the existence of steroids in an echinoderm, as well as characterizing key genes associated with the steroidogenic pathway. We propose that sex steroids might also be associated with the reproduction of H. scabra, and the identification of biosynthetic genes enables future functional studies to be performed.
Figure 1. Chromatograms and mass spectra corresponding to the LC–MS/MS analysis of estradiol, progesterone, and testosterone isolated from CNS and gonad tissue of H. scabra. (A) Representative chromatogram and MRM spectra for estradiol transitions m/z 271 → 145. (B) Representative chromatogram and MRM spectra for progesterone transitions m/z 315 → 109. (C) Representative chromatogram and MRM spectra for testosterone transitions m/z 289 → 97. STD; standard, CNS; central nervous system.
Figure 2. Schematic diagram showing the putative pathway for steroidogenesis in the H. scabra between mitochondria and the smooth endoplasmic reticulum (SER). In the pathway of steroid hormone biosynthesis, there are two major types of enzymes: cytochromes P450 (green) and other hydroxysteroid dehydrogenases (orange). In H. scabra, we identified gene transcripts encoding steroidogenic acute regulatory protein (StAR), cytochrome P450 10 (CYP10), 17alpha-hydroxylase/17, 20-lyase (CYP17), cytochrome P450 3A (CYP3A), 3beta-hydroxysteroid dehydrogenase (3β-HSD), and 17beta-hydroxysteroid dehydrogenase (17β-HSD). Question marks remain unclear. Steroidogenesis begins in the cells where cholesterol transport into the mitochondria facilitated by StAR. Once in the mitochondria, cholesterol may be converted to pregnenolone through the action of CYP10. Pregnenolone then diffuses to the SER, where it is further converted into a series of reactions to testosterone. Testosterone may be subsequently aromatized to estradiol through the action of CYP3A.
Figure 3. Characterization and phylogenetic tree analysis of H. scabra steroidogenic acute regulatory protein (StAR). (A) HscStAR contains the steroidogenic acute regulatory protein-related lipid transfer domain (START domain) (yellow highlight) which consists of cholesterol recognition/interaction amino acid consensus (CRAC) (L/V-(x)-Y-(x)-R/K) and dileucine- and tyrosine- (LL-(x)-Y) motifs (bold underlined letters), cleavage site (red double-underlined letters), start and stop codons (red letters). (B) Multiple sequence alignment of CRAC and LL-(x)-Y key motifs of StAR displays conservation among species. Sequence alignment represented by * = identical, : = strong homology, and . = less homology. (C) Phylogenetic tree of HscStAR constructed based on Neighbour-joining analysis with 1000 replicates bootstrap. Scale bar represents amino acid differences. Amino acid sequences and their accession number are shown in S2.
Figure 4. Characterization and phylogenetic tree analysis of H. scabra cytochrome P450 genes associated with steroidogenesis. (A) HscCYP10 contains the cytochrome P450 domain (yellow highlight) that includes a proline-rich motif (PPGTPITP), PERF/W, and KET/S(x)R(x)P(x)R regions (bold underlined letters). (B) HscCYP17 showed cytochrome P450 domain (yellow highlight) contains the heme-binding region (PFSTGRRVCLG) and PEHF region (bold underlined letters), and cleavage site (red double-underlined letters). (C) HscCYP3A contains cytochrome P450 domain (yellow highlight), which consists of PERF and heme-binding (PFGTGPRNCIG) regions (bold underlined letters), cleavage site (red double-underlined letters), and stop codons (red letters). (D) Phylogenetic tree of target CYP 450 involved in steroidogenesis constructed based on Neighbour-joining analysis with 1000 replicates bootstrap. Scale bar represents amino acid differences. Amino acid sequences and their accession number are shown in S2.
Figure 5. Characterization and phylogenetic tree analysis of H. scabra hydroxysteroid dehydrogenases (HSDs). (A) Hsc3β-HSD shows the start codons (red letters) and short-chain dehydrogenase/reductase (SDR) domain (yellow highlight) which contains NAD-binding motif (GGTGFIG) (bold underlined letters) (A). Shaded regions indicate motif sequences of amino acids identical to NAD-binding motif. (B) Hsc17β-HSD contains the short-chain dehydrogenase/reductase (SDR) domain (yellow highlight) that composed of 2 important motifs; NNAG and YxxxK (bold underlined letters), cleavage site (red double-underlined letters), and stop codons (red letters). (C) Phylogenetic tree of target HSDs involved in steroidogenesis constructed based on Neighbour-joining analysis with 1000 replicates bootstrap. Scale bar represents amino acid differences. Amino acid sequences and their accession number are shown in S2.
Figure 6. Tissue distribution and quantitative analysis of target genes involved in steroidogenesis in H. scabra. (A) Left—Schematic of sea cucumber showing tissues analysed by RT-PCR. Right—Agarose gel showing amplicons for StAR; steroidogenic acute regulatory protein, CYP10; cytochrome P450 10, CYP17; 17α-hydroxylase/17,20-lyase, CYP3A; cytochrome P450 3A, 3β-HSD; 3β-hydroxysteroid dehydrogenase, 17β-HSD; 17β-hydroxysteroid dehydrogenase, 16S rRNA; internal control, and negative; no RT-template. (B) Quantitative expression profiles in gonadal tissues of H. scabra during reproductive maturation (stages 1–5) (n = 10 for each sex at each stage). Data were normalized against 16S rRNA and the relative expression levels represented by the mean ± SD. Different letters indicate significant difference (p < 0.05) among reproductive stages of females (capital letters) and males (small letters) H. scabra.
Figure 7. Effect of estradiol and progesterone on Vtg expression in ovarian tissue explants between 0 and 180 min. Relative gene expression levels of Vtg shown by quantitative real time-PCR (n = 3). Data were normalized against 16S rRNA and represented as mean ± S.D. Different letters indicate statistically significant different (p < 0.05) among the groups at the same incubation time.
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