Gnrh3 Regulates PGC Proliferation and Sex Differentiation in Developing Zebrafish

Area of Science:

• Reproductive biology
• Developmental biology
• Endocrinology

Background:

• Gonadotropin-releasing hormone (Gnrh) is essential for reproduction, controlling luteinizing hormone release, ovulation, and sperm release.
• Gnrh acts as a decapeptide, playing a vital role in the reproductive processes of numerous species.

Purpose of the Study:

• To investigate a novel role for Gnrh3 beyond its known reproductive functions.
• To explore the impact of Gnrh3 deficiency on primordial germ cell (PGC) development and early sex differentiation in zebrafish.

Main Methods:

• CRISPR/Cas9 gene editing was used to generate Gnrh3-null zebrafish (gnrh3-/-).
• Quantification of PGCs, assessment of PGC proliferation and apoptosis (TUNEL assay), and analysis of signaling pathways (AKT, ERK1/2 phosphorylation).
• Pharmacological interventions including Gnrh analog (Alarelin) treatment and MEK inhibitor (PD0325901) administration, alongside gene expression analysis (sox9a, amh, cyp11b, cyp19a1a).

Main Results:

• Gnrh3-null zebrafish exhibited a male-biased sex ratio and reduced PGC numbers post-dome stage, with no increased PGC apoptosis.
• PGC proliferation was significantly impaired in gnrh3-/- embryos, evidenced by a lack of detectable proliferating PGCs.
• Gnrh3 deficiency led to decreased ERK1/2 phosphorylation, which was rescued by Alarelin treatment and blocked by PD0325901, indicating a MAPK-dependent mechanism.
• Upregulation of male sex differentiation markers (sox9a, amh, cyp11b) and downregulation of the female marker (cyp19a1a) were observed in gnrh3-/- zebrafish.

Conclusions:

• Gnrh3 plays a critical role in early sex differentiation in zebrafish.
• Gnrh3 regulates PGC proliferation, a key process influenced by the MAPK signaling pathway.
• These findings reveal a previously unrecognized function of Gnrh3 in germ cell development and sex determination.