Theoretical Analysis and Expression Profiling of 17β-Hydroxysteroid Dehydrogenase Genes in Gonadal Development and Steroidogenesis of Leopard Coral Grouper (Plectropomus leopardus)

Abstract

The differentiation and developmental trajectory of fish gonads, significantly important for fish breeding, culture, and production, has long been a focal point in the fields of fish genetics and developmental biology. However, the mechanism of gonadal differentiation in leopard coral grouper (Plectropomus leopardus) remains unclear. This study investigates the 17β-Hydroxysteroid Dehydrogenase (Hsd17b) gene family in P. leopardus, with a focus on gene characterization, expression profiling, and functional analysis. The results reveal that the P. leopardus's Hsd17b gene family comprises 11 members, all belonging to the SDR superfamily. The amino acid similarity is only 12.96%, but conserved motifs, such as TGxxxGxG and S-Y-K, are present in these genes. Hsd17b12a and Hsd17b12b are unique homologs in fish, and chromosomal localization has confirmed that they are not derived from different transcripts of the same gene, but rather are two independent genes. The Hsd17b family genes, predominantly expressed in the liver, heart, gills, kidneys, and gonads, are involved in synthesizing or metabolizing sex steroid hormones and neurotransmitters, with their expression patterns during gonadal development categorized into three distinct categories. Notably, Hsd17b4 and Hsd17b12a were highly expressed in the testis and ovary, respectively, suggesting their involvement in the development of reproductive cells in these organs. Fluorescence in situ hybridization (FISH) further indicated specific expression sites for these genes, with Hsd17b4 primarily expressed in germ stem cells and Hsd17b12a in oocytes. This comprehensive study provides foundational insights into the role of the Hsd17b gene family in gonadal development and steroidogenesis in P. leopardus, contributing to the broader understanding of fish reproductive biology and aquaculture breeding.

Keywords: 17β-Hydroxysteroid Dehydrogenase; Plectropomus leopardus; gonadal development; sex differentiation; steroidogenesis.

Figure 1

Phylogenetic tree (A) and gene copy number (B) analysis of the Hsd17b gene family in P. leopardus and other species. Note: The phylogenetic tree was constructed by MEGA 11.0 using the neighbor joining method, and the confidence values higher than 70 were indicated on the tree.

Figure 2

Gene structure (A), motif analysis (B), and conserved domains (C) of the Hsd17b gene family in P. leopardus. Note: In (B), red boxes represent Hsd17b genes belonging to Clade I; yellow boxes represent Hsd17b genes belonging to Clade II; green boxes represent Hsd17b genes belonging to Clade III; black boxes represent Hsd17b genes belonging to Clade IV; blue boxes represent Hsd17b genes belonging to Clade V. Boxes with different colors indicated different motifs and domains.

Figure 3

Subcellular localization of the Hsd17b gene family in P. leopardus.

Figure 4

Chromosomal distribution (A) and protein–protein interaction analysis (B,C) of the Hsd17b gene family in P. leopardus. Note: (B) original network, (C) network clustered to 6 clusters.

Figure 5

Expression profiles of the Hsd17b gene family in P. leopardus across different developmental stages (A) and all tissues (B). Note: 120 dph: 120 days post-hatch, the gonadal undifferentiated stage; BiT: 15-month-old testis in the bisexual phase; BiO: 15-month-old ovary in the bisexual phase; 3YT: 3-year-old testis; 3YO: 3-year-old ovary. Red, high expression. The color scale indicated the TPM values. The colors from blue to red represent the lower TPM values to higher TPM values.

Figure 6

Localization of Hsd17b4 and -12a RNA by fluorescence in situ hybridization (FISH) and histology. Note: (Aa-1–Aa-5) includes stages of 120 days post-hatch (120 dph), bisexual testis (BiT) phase, bisexual ovary (BiO) phase, 3-year-old males (3YT), and 3-year-old females (3YO) with HE staining of gonads (20× magnification). Gonad tissue from the P. leopardus in the BiT and BiO stages was used for localizing Hsd17b4, Hsd17b12a, and Vasa RNA via FISH experiments in (B) (40× magnification). Stain all DNA blue (b-1,c-1,d-1,e-1,f-1,g-1) with diamidino-phenyl-indole (DAPI). Digoxigenin (DIG) stains green (Vasa: b-3,c-3; Hsd17b12a: d-3,e-3,f-3,g-3) and biotin (BIO) stains red (Hsd17b4: b-2,c-2,f-2,g-2; Vasa: d-2,e-2). Merge is a merged image (b-4,c-4,d-4,e-4,f-4,g-4). Staining was analyzed by confocal microscopy. Gscs: germ stem cells; I, early previtellogenic phase; II, late previtellogenic phase; III, early vitellogenic phase; IV, fully vitellogenic phase; Oc: oocyte; Psc: primary spermatocyte phase; Sc, spermatocytes; St, spermatids. The scale bar is located at the bottom right corner of the figure, representing 50 μm.