Prolactin in the brushtail possum (Trichosurus vulpecula): development of homologous radioimmunoassay using recombinant possum prolactin.
We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.
Crawford
J L
JL
Reproduction Group, AgResearch Ltd., Wallaceville Animal Research Centre, Upper Hutt, New Zealand. janet.crawford@agresearch.co.nz
Lun
S
S
Demmer
J
J
Eckery
D C
DC
D000818:Animals; D001681:Biological Assay / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D000077465:Cabergoline; D018491:Dopamine Agonists / Q000494:pharmacology; D004873:Ergolines / Q000494:pharmacology; D005260:Female; D006107:Granulosa Cells; D008297:Male; D009520:New Zealand; D009893:Opossums / Q000378:metabolism / Q000502:physiology*; D011374:Progesterone / Q000032:analysis; D011388:Prolactin / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011863:Radioimmunoassay / Q000379:methods / Q000662:veterinary*; D011869:Radioligand Assay / Q000662:veterinary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D018491:Dopamine Agonists; D004873:Ergolines; D011994:Recombinant Proteins; D011374:Progesterone; D013973:Thyrotropin-Releasing Hormone; D012313:RNA; D011388:Prolactin; D000077465:Cabergoline