Transcriptomic insights into gametogenesis and steroidogenic enzyme gene regulation in Australasian snapper (Chrysophrys auratus) gonads during the reproductive cycle

Technical Summary: Transcriptomic Insights into Gametogenesis and Steroidogenic Enzyme Gene Regulation in Australasian Snapper (Chrysophrys auratus) Gonads

Problem Statement
The Australasian snapper (Chrysophrys auratus) is a priority species for aquaculture diversification in New Zealand. While the reproductive physiology of snapper has been characterized phenotypically, the molecular mechanisms driving gametogenesis and the specific gene expression dynamics throughout the reproductive cycle remain unexplored. Unlike its close relative, the red seabream (Pagrus major), which has a well-established breeding industry, snapper lacks a comprehensive transcriptomic resource. Furthermore, the regulatory controls of sex steroid biosynthesis, particularly the role of gonadal aromatase (cyp19a1a) in relation to plasma estradiol-17β (E2) levels, are unknown in this species. This study addresses the gap in molecular knowledge required to advance broodstock management and understand sexual differentiation in captive snapper.

Methodology
The study utilized a cohort of 84 captive-bred snapper (42 females and 42 males) sampled throughout their maiden spawning cycle under ambient photoperiod and temperature conditions. Gonadal tissue was collected at specific developmental stages:

• Females: Five stages of oogenesis (F1: immature; F2: resting post-spawning; F3: developing; F4: developed/vitellogenic; F5: spawning).
• Males: Three stages of spermatogenesis (M2: resting post-spawning; M3: developing; M4: developed). Note: M1 (immature) was not sampled due to limitations.

Experimental Workflow:

1. RNA Sequencing: Total RNA was extracted from gonadal tissues and sequenced on the Illumina NovaSeq platform to generate 150 bp paired-end reads.
2. Genome Annotation: The authors improved the existing snapper genome annotation using the BRAKER3 pipeline, integrating RNA-seq data from brain, pituitary, and gonad tissues with protein evidence from OrthoDB. Liftoff was used to map annotations from the yellowfin seabream (Acanthopagrus latus) to the snapper genome. OrthoFinder was employed to identify orthologous groups using zebrafish (Danio rerio) as a reference.
3. Quantification and Differential Expression: Gene expression was quantified using the nf-core/rnaseq pipeline (STAR and Salmon). Differential expression analysis (DEA) was performed using DESeq2 to identify sex-biased genes and stage-specific differentially expressed genes (DEGs) between gametogenic stages.
4. Functional Analysis: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using clusterProfiler to interpret biological processes associated with DEGs.
5. Target Gene Analysis: Specific steroidogenic enzymes (e.g., star, cyp11a1.2, cyp17a1, hsd3b1, cyp19a1a, cyp11b1, hsd11b2) and hormone receptors (fshr, lhcgr, ar, esr1, esr2a) were analyzed individually using normalized gene counts and statistical testing (ANOVA or Kruskal-Wallis with post-hoc corrections).

Key Results

• Genome Annotation: The study successfully annotated 26,418 protein-coding genes in the snapper genome, with 97.8% of BUSCOs identified as complete. This resource provides a foundation for future genomic studies.
• Sex-Biased Expression: Transcriptome profiling revealed distinct clustering of male and female gonads. There were 3,588 upregulated DEGs in females and 3,938 in males.
  • Females: The number of upregulated DEGs increased as oogenesis progressed, peaking during the spawning stage (F5). Functional enrichment highlighted upregulation of ribosomal processes and metabolism-associated genes.
  • Males: The highest number of upregulated DEGs occurred in the early developing (M3) and developed (M4) stages. Enrichment analysis indicated upregulation of cell motility and morphogenesis-related genes, consistent with spermiogenic processes.
• Steroidogenic Enzyme Expression:
  • In females, most genes involved in testosterone synthesis (star, cyp11a1.2, cyp17a1, hsd3b1) increased significantly as oogenesis progressed, peaking at spawning.
  • Notable Exception: The gonadal aromatase gene, cyp19a1a, did not show significant variation in expression across any gametogenic stages in females, despite high overall expression levels. This contrasts with the known fluctuation of plasma E2 levels in snapper and the expression patterns of cyp19a1a in red seabream.
  • In males, expression patterns were less regular. cyp11b1 (involved in 11-KT synthesis) was downregulated in the developed stage (M4), while hsd11b2 was upregulated in the resting post-spawning stage (M2).
• Hormone Receptors:
  • fshr (FSH receptor) was highly expressed in males compared to females.
  • In females, lhcgr and esr1 expression increased throughout the cycle, peaking at spawning (F5).
  • The androgen receptor (ar) was expressed at higher levels in females overall compared to males.

Significance and Claims
The authors claim that this study provides the first transcriptome-wide exploration of snapper gonads throughout a reproductive cycle. The primary contributions are:

1. Resource Generation: The improved genome annotation (26,418 genes) serves as a critical tool for future studies on snapper, extending beyond reproductive research.
2. Molecular Insights: The identification of sex-biased gene expression and stage-specific DEGs offers a molecular basis for understanding gametogenesis. The authors suggest these profiles could potentially be used to differentiate sexes in juvenile snapper prior to sex inversion, though this is proposed as a future application of the data rather than a demonstrated result.
3. Regulatory Mechanism Discovery: The study challenges the assumption that cyp19a1a expression drives E2 fluctuations in snapper, as seen in other species. The constant expression of cyp19a1a despite rising E2 levels suggests that E2 synthesis in snapper may be regulated by precursor availability (e.g., testosterone or estrone) or post-transcriptional mechanisms rather than transcriptional control of the aromatase gene.
4. Aquaculture Utility: By characterizing the transcriptomic landscape of captive reproduction, the study lays the groundwork for developing methods to control reproductive timing and sexual differentiation in aquaculture settings, addressing a key bottleneck in snapper domestication.

The paper maintains a modest tone regarding future implications, noting that the bulk RNA-seq approach limits the ability to resolve cell-specific expression shifts (e.g., within specific follicle stages) and that the highly asynchronous nature of snapper spermatogenesis complicates the isolation of specific molecular events at the single-cell level. The authors suggest that future work utilizing single-cell RNA sequencing or follicle separation techniques would be necessary to further elucidate these mechanisms.