The transforming growth factor‑β (TGF‑β) superfamily plays critical roles in maintaining organismal homeostasis and regulating development. TGF‑β ligands are secreted extracellularly as homodimers or heterodimers and bind to tetrameric receptor complexes consisting of two type‑II and two type‑I receptors. Type‑II receptors mediate the phosphorylation of type‑I receptors, which subsequently transduce downstream signals through the Smad pathway. Anti‑Müllerian hormone (Amh) and gonadal somatic cell‑derived factor (Gsdf) are evolutionarily conserved TGF‑β ligands in vertebrates. Notably, the gsdf gene has been lost in tetrapods and is retained exclusively in fish. Both molecules participate in key developmental processes, including the regulation of germ‑cell homeostasis, sex determination, and sex differentiation.

Previous studies have demonstrated that loss‑of‑function mutations in amh or gsdf in fish disrupt the balance between germ‑cell proliferation and differentiation, triggering excessive proliferation and blocked differentiation of early germ cells, which ultimately causes gonadal hypertrophy. These observations indicate that maintenance of germ‑cell homeostasis is a conserved shared function of Amh and Gsdf. Nevertheless, their functional distinctions and the molecular basis underlying their synergistic crosstalk remain poorly understood.

Recently, a research team led by Prof. GUI Jianfang from the Institute of Hydrobiology (IHB) of the Chinese Academy of Sciences uncovered a novel synergistic regulatory mechanism for two core TGF‑β superfamily ligands — Amh and Gsdf — which control germ‑cell homeostasis through functional heterodimerization in gibel carp (Carassius gibelio). This work has been published in Science Bulletin.

Using gynogenetic gibel carp (Carassius gibelio) as the experimental model, the group dissected the biological functions and cooperative mechanisms of Amh and Gsdf in germ‑cell homeostasis. Combining CRISPR/Cas9 mutagenesis, histological examination, immunofluorescence staining, multiple molecular‑biological approaches, protein‑protein interaction and structural analysis, luciferase reporter assays, protein‑structure prediction, ex‑vivo gonadal culture, and in‑vivo protein‑rescue experiments, the authors deciphered the molecular mechanism whereby Amh and Gsdf assemble into functional heterodimers to synergistically sustain germ‑cell homeostasis via the Bmpr2a/Bmpr1bb‑Smad1/5/8‑Inha signaling cascade. They further explored the evolutionary conservation of this heterodimer‑driven regulatory mode among teleost fishes.

The authors generated amh and gsdf single‑knockout mutants, as well as amh;gsdf double mutants using CRISPR/Cas9 technology.Disruption of either gene perturbs the germ‑cell proliferation‑differentiation balance and induces ovarian hypertrophy. Double mutants exhibit more severe gonadal abnormalities than either single mutant, confirming synergistic activities between Amh and Gsdf.

Results from ex‑vivo gonadal culture and in‑vivo protein‑rescue assays revealed that both Amh/Gsdf heterodimers and their respective homodimers signal through the Bmpr2a/Bmpr1bb‑Smad1/5/8‑Inha cascade. The Amh/Gsdf heterodimer exhibits superior signaling potency and biological efficacy compared with Amh and Gsdf homodimers, following the functional‑activity hierarchy: Amh/Gsdf heterodimer > Amh homodimer > Gsdf homodimer.

Meanwhile, protein‑protein interaction and structural analyses demonstrated that Amh assembles functional heterodimers with Gsdf through contacts between their pro‑domains and disulfide‑bond‑linked mature domains, both in vitro and in gibel carp ovaries in vivo. Recombinant Amh‑Gsdf heterodimer protein can efficiently rescue the hypertrophic ovarian phenotype observed in amh‑deficient mutants.

The ability to form a potent Amh/Gsdf heterodimer was also found to be evolutionarily conserved. In vitro experiments using proteins from zebrafish and medaka — two widely studied model fish — showed the same pattern: the heterodimer activated downstream signalling more efficiently than either homodimer. Notably, amh is present in all jawed vertebrates, while gsdf exists in most jawed vertebrates except tetrapods. The strong selection for gsdf retention in fish may reflect the evolutionary advantage conferred by the more robust heterodimeric control of germ cell homeostasis, which helps maintain the high fecundity of fish species.

The findings provide novel insights into the mechanisms underlying the proliferation differentiation balance of massive germ cells in fish species, and open new possibilities for precise control of gonadal development in aquaculture species.

(Editor: MA Yun)