Molecular cloning of the cDNAs for pituitary glycoprotein hormone α subunits of two species of duck and their gene regulation

The cDNAs encoding pituitary glycoprotein hormone a subunits (PGHas) of two species of duck (Muscovy duck, Cairina moschata and Pekin duck, Anas platyrhynchos domesticus) were cloned and sequenced to better understand the phylogenic diversity and evolution of PGH alpha molecules in vertebrates. Oligonucleotide primers were designed and used for reverse transcription PCR (RT-PCR) amplification of PGH alpha cDNA fragments from total cellular RNA of pituitary glands. The remaining sequences were completed by rapid amplification of the cDNA ends. The nucleotide sequence of isolated PGH alpha cDNA of both ducks are identical, including 81 bp of 5' untranslated region (UTR), 360 bp of coding region, and 272 bp of 3'-UTR followed by a 13 bp poly(A)(+) tract. The total number of amino acids deduced from the cDNA of the duck PGHa is 120 with a signal peptide of 24 amino acids and a mature protein of 96 amino acids. PGH alphas of the ducks (order Anseriformes) share 96% homology of amino acid sequence in signal peptide, and 100% homology in mature proteins with chicken, quail and turkey (order Galliformes). Our data thus demonstrate identical inter-order homology of PGH alpha mature protein in birds. Ten cysteine residues, presumably forming five disulfide bonds within the a subunit, and four proline residues, presumably responsible for folding of the molecule, are conserved in the alpha subunit of ducks. Northern blot analysis revealed that PGHa mRNA is expressed only in the pituitary. In order to study factors regulating the gene expression of PGH alpha mRNA, duck pituitary fragments were incubated with GnRH, TRH, testosterone, or triiodothyronine (T-3). GnRH and TRH increased, while testosterone and T-3 decreased, PGH alpha mRNA levels. This is the first report in birds of TRH up-regulation and down-regulation by testosterone and T-3 under in vitro conditions. The present study demonstrates both ducks have the same cDNA nucleotide and deduced amino acid sequences in the PGH alpha subunit, exhibiting identical inter-genus homology within the family of Anatidae. The findings from mRNA expression work suggest that hypothalamic GnRH and TRH up-regulate, while testosterone and T-3 down-regulate, PGH alpha gene expression in ducks.