Anti-Müllerian hormone (Amh) is in mammals known as a TGFβ type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis.

The crystal structure of an anionic form of salmon trypsin has been determined at 1.82 A resolution. We report the first structure of a trypsin from a phoikilothermic organism in a detailed comparison to mammalian trypsins in order to look for structural rationalizations for the cold-adaption features of salmon trypsin. This form of salmon trypsin (ST II) comprises 222 residues, and is homologous to bovine trypsin (BT) in about 65% of the primary structure. The tertiary structures are similar, with an overall displacement in main chain atomic positions between salmon trypsin and various crystal structures of bovine trypsin of about 0.8 A. Intramolecular hydrogen bonds and hydrophobic interactions are compared and discussed in order to estimate possible differences in molecular flexibility which might explain the higher catalytic efficiency and lower thermostability of salmon trypsin compared to bovine trypsin. No overall differences in intramolecular interactions are detected between the two structures, but there are differences in certain regions of the structures which may explain some of the observed differences in physical properties. The distribution of charged residues is different in the two trypsins, and the impact this might have on substrate affinity has been discussed.

A leukocyte cDNA library from Atlantic salmon, based on oligo-dT priming, was constructed in lambda-gt10. Immunoglobulin heavy chain (IgH) cDNA were isolated from the library using a specific probe generated by polymerase chain reaction (PCR) between two conserved areas within the variable region (second and fourth frame region). Two cDNA clones encoding the entire constant region of membrane-bound IgH, and one cDNA encoding the entire constant region of secretory IgH were sequenced, revealing messages from two isotypic IgM genes. Both genes were shown to be present in haploid embryos and have been isolated from a genomic library, the exons and deduced amino acid sequences of which are presented here (salmon CHA and CHB). The splicing of transcripts encoding the membrane-bound IgH excises the whole fourth exon as in other teleosts. The nucleotide and amino acid identity between salmon CHA and CHB are 98.2%, and 96.2%, respectively. Two subfractions of IgM from Atlantic salmon separated by ion chromatography can be explained by a net exchange of basic residues in salmon CHB compared to CHA. The finding of two closely related salmon CH genes is in accordance with the quasi-tetraploid state of the Atlantic salmon genome.

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.