RESUMO
A partial prolactin (PRL) cDNA was specifically PCR amplified from a cDNA library constructed from pituitary mRNAs of the newt (Cynops pyrrhogaster) and cloned into plasmid vectors. One clone thus obtained contained a 739-bp insert encoding the C-terminal amino acid sequence of the mature hormone molecule. Using this clone as a probe, the full-length newt PRL cDNA was screened from the cDNA library. The PRL cDNA clone thus obtained consisted of 1024 bp encoding the entire sequence of the mature PRL molecule in addition to its signal peptide. The amino acid sequence of newt PRL deduced from its nucleotide sequence showed higher homologies with those PRL sequences of tetrapod animals than with those of teleosts. Northern blot analysis revealed the newt PRL mRNA size to be approximately 1 kb. In situ hybridization using the newt PRL cDNA as a probe revealed that the pituitary region expressing PRL mRNA corresponded to that immunoreactive with antiserum against PRL. PRL mRNA levels in the pituitary of newts subjected to room and low temperatures were determined by Northern analysis employing the PRL cDNA as a probe. PRL mRNA levels were significantly higher in the pituitaries of newts subjected to 10 degrees than in those of newts kept at 23 degrees. Likewise, immunoassayable plasma PRL levels were higher in animals subjected to 10 degrees than in those kept at 23 degrees.
Assuntos
DNA Complementar/biossíntese , Prolactina/biossíntese , RNA Mensageiro/biossíntese , Salamandridae/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , DNA Complementar/genética , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Hipófise/metabolismo , Plasmídeos/genética , Prolactina/química , Prolactina/genética , Radioimunoensaio , TemperaturaRESUMO
The amino acid sequence of newt (Cynops pyrrhogaster) prolactin deduced from the nucleotide sequence of its cDNA showed a relatively high homology with sequences of chicken and sea turtle prolactins as well as with those of anuran prolactins. Cynops prolactin receptor transcripts were detected in various tissues and organs, suggesting that prolactin plays multiple roles in urodeles. Urodele prolactin was purified from the pituitaries of C. pyrrhogaster. Antiserum against this prolactin was used for radioimmunoassay of plasma prolactin and immunoneutralization experiments. Endogenous prolactin was shown to induce migration to water, courtship behavior, and cessation of spermatocytogenesis in the Cynops newt. The hormone was found to be involved in the development of cloacal glands such as the lateral and abdominal glands, growth of the tail and Mauthner neurons, secretion of oviducal jelly, and enhanced synthesis of a female attracting pheromone (sodefrin), and responsiveness of the olfactory epithelium to sodefrin. In most of these cases, prolactin was found to act synergistically or antagonistically with sex steroids. We also discovered that hypersecretion of prolactin in the newts subjected to cold temperature was induced by hypothalamic stimulation rather than release from hypothalamic inhibition.
