Isolation, characterization and radioimmunoassay of luteinizing hormone in the brushtail possum

Abstract

Luteinizing hormone (LH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands. The purification procedure consisted of ammonium sulfate precipitation followed by triazinyl-dye chromatography, hydrophobic interaction chromatography and gel filtration. A yield of 10 µg LH g^-1 pituitary with a recovery of 20% was obtained from 1400 pituitary glands (20·3 g). Contamination with possum follicle-stimulating hormone (FSH) was ≤0.05%. The amino acid analysis and the N-terminal sequencing for 10 cycles revealed close homology with LH from other mammals. Minor amounts of LH that had been truncated near the N-terminal were also detected. No contaminating proteins were found by amino acid sequencing. The potency of possum LH was 20% that of ovine LH in a receptor assay using possum testicular receptors and 4% that of ovine LH when bovine corpora lutea receptors were used. Possum LH was able to stimulate production of cyclic adenosine 3′ ,5′-monophosphate by bovine granulosa cells. A radioimmunoassay (RIA) for possum LH using ¹²⁵I-possum LH and an antiserum raised against ovine LH was developed. The RIA has a sensitivity of 0·15 ng mL^-1 , a 50% displacement of 1·9 ng mL^-1 and a cross-reactivity of <0 · 02% against possum FSH. Plasma concentrations were 0·24 ± 0· 04 ng mL^-1 (n = 8) and 0·39 ± 0·12 ng mL^-1 (n = 8) in female and male possums respectively. Administration of mammalian gonadotrophin-releasing hormone (GnRH) and chicken GnRH II stimulated increases in plasma LH concentrations in male and female possums. When comparing LH responses with administration of mammalian GnRH or chicken GnRH II, plasma LH concentrations remained elevated for a longer period of time in males than in females (P < 0· 01); plasma LH concentrations also remained elevated for longer after mammalian GnRH than after chicken GnRH II (P < 0· 01). Gonadectomy stimulated an increase in plasma concentrations of LH in both male (P < 0· 01) and female (P < 0· 05) possums. The rate of increase in plasma LH concentrations in males was faster than that in females. In summary, we have purified, partially characterized, and developed a RIA for possum LH.