Introduction
Effective control of reproduction is fundamental for sustainable aquaculture of farmed species, and understanding in-depth the molecular regulatory systems governing gametogenesis becomes essential. In vertebrates, t he brain-pituitary-gonad (BPG) axis regulates reproduction though gonadotropins, sex steroids, and local gonadal factors , t he latter being largely unknown in fish. Among these, some members of the transforming growth factor β (TGF-β ) family, including the Anti-Müllerian Hormone (Amh) and the Gonadal Soma-Derived Factor (Gsdf), are emerging as key regulators of early stages of gametogenesis and sex determination and differentiation [1] . To date, t heir characterization links them to male sex differentiation [2,3] and to the proliferation of type A spermatogonia (SgA) [4,5] , while their regulatory roles in male and female adult gonads remains to be clarified . The European sea bass (Dicentrarchus labrax), an economically important species in Mediterranean aquaculture, provides a robust experimental model for this research based on the good number of tools available for reproductive physiology studies . Based on our previous knowledge and developed tools (sea bass recombinant proteins and specific antibodies) [6,7], t he aim of this work is the functional characterization of Amh and Gsdf in male and female sea bass during puberty and adult gametogenesis.
Materials and methods
To further investigate on these gonadal factors, a multidisciplinary approach including molecular, biochemical and physiological analysis was performed . Recombinant sea bass Amh and Gsdf produced in CHO cells were used to treat in vitro cultured testes and ovaries to assess their direct effects on the gonads. Specific antibodies allowed protein detection in gonadal extracts and their localization in histological sections to disclose the role of these proteins in the local control of gametogenesis. In vivo administration of hormone-coding plasmids were conducted in the pre-meiotic stage animals to analyse their action at the organism level. Finally, transactivation assays were done in Cos7 cells f or studying Amh and Gsdf intracellular signalling pathways, and to identify the specific receptor for Gsdf, which remains unknown .
Results
Gene expression: During juvenile gonadal differentiation , expression levels of amh , amhr2 , gsdf1 and gsdf2 peaked at 250 d ays post-hatch. In testis of 1-year-old males that enter precocious puberty, t he expression of gsdf1 and gsdf2 decreased compared with their siblings that remained immature. In adult males, gsdf1 , and to a lesser extent gsdf2 , showed maximum expression levels in premeiotic testis, while i n adult females, the expression levels of gsdf genes were lower than in testis, and gsdf2 mRNA predominated in post-ovulatory ovaries and in isolated follicular cells.
Protein localization : In adult testis, Amh was immunodetected in Sertoli cells surrounding early germ cell generations, whereas Gsdf was localized in Sertoli cells surrounding SgA but exclusively in the central region of each lobule. In ovaries, Amh was present in follicular cells of both early and late-vitellogenic stages , while Gsdf presence was confined to pre-vitellogenic follicular cells. In addition, Amhr2 was detected in SgA of post-spawning and pre-meiotic testis and in follicular cells of pre-/vitellogenic ovaries.
Functional analysis : In vitro cultured pre-meiotic testis or pre-/early-vitellogenic ovaries treated with recombinant Amh revealed its steroidogenic activity, resulting in an increase of steroid production. In the same vein, in vivo intramuscular injection of Amh expression plasmid caused an increase of circulating sexual steroids. Transactivation experiments in Cos7 cells confirmed that, in addition to the canonical Smad pathway, the sea bass Amh/Amhr2 pair was able to signal through the main steroidogenic cAMP pathway and its action was inhibited by the cAMP antagonist Rp-cAMPS . Finally, all the sea bass TGF-β type I receptor genes were cloned and functionally tested in Cos7 cells in combination with type II receptors to define the precise intracellular mechanism of action of Amh and to find the specific Gsdf receptor.
Discussion and Conclusion
The novel steroidogenic activity of Amh found in our experiments in sea bass indicates that fish Amh may act differently from mammalian Amh, which inhibit Fsh-induced on steroidogenesis. Gene expression analysis indicates that Gsdf1/2 and Amh have a clear function in male gonad differentiation. Moreover, the gonadal expression of gsdf genes during the adult reproductive cycle suggests a predominant role in adult males rather than females. The gsdf1 downregulation in precociously maturing males suggests its potential as an early marker and target for puberty regulation. Protein detection and cellular localization demonstrated a paracrine role of both factors in germ cell proliferation, in the onset of spermatogenesis before meiosis, in folliculogenesis, and during vitellogenesis. Finally, the description of the specific interactions between type II and type I receptors, and their particular signaling trough phosphorylation of Smad 2/3 or Smad 1/5/8 proteins contributes valuable insights into the TGFβ signalling network in teleost gonadal function.
Funding
Funded by MCIN/AEI/10.13039/501100011033/ and by ERDF a way of making Europe (PID2021-122929OB-C32) . GV supported A.M. PhD contract (GRISOLIAP/2020/129).
References