FOLLICLE-STIMULATING-HORMONE IN THE BRUSHTAIL POSSUM (TRICHOSURUS-VULPECULA) - PURIFICATION, CHARACTERIZATION, AND RADIOIMMUNOASSAY

Follicle-stimulating hormone (FSH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands by using the following purifi cation techniques: fractional ammonium sulfate precipitation, triaziny l-dye affinity chromatography, hydrophobic interaction chromatography, and gel filtration. A yield of 18 mu g of FSH per gram of pituitary, with a recovery of 12%, was obtained from 1400 glands (20.3 g wet weig ht). The purified FSH activity per gram of protein was 1320 times more potent than the initial pituitary homogenate. Contamination with poss um luteinizing hormone (LH) was <0.02%. The amino acid composition of possum FSH was similar to that of ovine FSH. Amino-terminal sequencing for 11 cycles indicated that the alpha subunit has the same sequence as ovine FSH except for residue 7, where the possum FSH alpha subunit contains isoleucine compared to the ovine subunit which contains threo nine. The beta subunit has two substitutions in the first 11 residues and does not contain the N terminal serine that is found in ovine FSH. Amino acid sequencing did not detect any contaminating proteins. Poss um FSH bound possum and bovine testicular receptors with similar affin ities. It was also able to stimulate in vitro cAMP production by Chine se hamster ovary cells which express recombinant FSH receptors. In the receptor assays and the bioassay possum FSH has about 21% of the pote ncy of ovine FSH (USDA-oFSH-19-SIAFP-RP2). An RIA was developed for po ssum FSH using I-125-possum FSH and an antiserum raised against human FSH. The RIA has a sensitivity of 0.3 ng/ml, a 50% displacement of 2.7 ng/ml, and a cross reactivity of 0.05% against possum LH. Plasma FSH levels in male possums (10.4 +/- 2.4 ng/ml, n = 4) were higher (P < 0. 05) than levels in females (1.0 +/- 0.1 ng/ml, n = 4). Five days after gonadectomizing these possums the plasma FSH levels increased (P < 0. 05) to 27.1 +/- 0.2 ng/ml in the males and to 6.6 +/- 2.0 ng/ml in the females. In summary, we have purified and partially characterized pos sum FSH. We have also set up an RIA for the hormone and shown that mal es have higher levels than females and that plasma FSH increases after gonadectomy. (C) 1997 Academic Press.