Effects of different batches of 125iodine on properties of 125I-hFSH and characteristics of radioligand-receptor assays.

Radioiodination of highly purified human follicle-stimulating hormone (hFSH) (4000 IU/mg) was performed every other week for 23 weeks using 2 mCI carrier free Na125I (Amersham Corp., 15 mCi/micrograms I2) in the presence of lactoperoxidase. Incorporation of 125I into hFSH was determined by the method of R. C. Greenwood, W. M. Hunter, and J. S. Grover (1963) Biochem. J. 89, 114). Hormone binding was studied in vitro under steady-state conditions (16 h, 20 degrees C) using different calf testis membrane preparations having similar receptor characteristics. Each 125I-hFSH preparation was characterized for maximum bindability, specific activity of bindable radioligand as determined by self-displacement analysis, and by determination of Ka and Rt. Incorporation of 125I into FSH was relatively constant over the large number of experiments (62.4 +/- 6.4 microCi/micrograms; n = 23). By comparison, however, specific radioactivity of the receptor bindable fraction of 125I-hFSH was related to the lot of 125I utilized, and was significantly (P less than or equal to 0.01) lower and more variable (28.7 +/- 10.5 microCi/micrograms). Maximum bindability of 125I-hFSH was not correlated to specific activity (r = 0.06) but was negatively correlated to hFSH 125I incorporation (r = -0.47; P less than or equal to 0.05). These observations demonstrate the need to assess the quality of each batch of radioligand before undertaking radioligand-receptor assays and suggest that differences in Na125I lots affect specific radioactivity of the radioligand and its receptor binding characteristics.

Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides.

Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor.

Elevation of serum testosterone during chronic LHRH agonist treatment in the bull.

The objective of this study was to try to depress serum testosterone (T) in bulls by prolonged treatment with a potent luteinizing hormone-releasing hormone (LHRH) agonist. Eight sexually mature bulls (325 to 475 kg) were assigned to treatment or control groups. Treatment consisted of 150 micrograms nafarelin acetate 6-D-2-naphthyl-alanine-LHRH (LHRH-A) injected im every 6 h for 15 d. Bovine serum albumin (BSA, .01%) in a carrier solution was injected at the same times in control bulls. Serial 15-min blood samples were collected via jugular cannula during the initial 36 h of treatment and during 6-h windows on d 4, 8 and 14. Bulls were slaughtered and pituitaries and testes collected on d 15. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and T were elevated after initial injection of LHRH-A, but returned to basal concentrations by 12, 5 and 17 h, respectively. Prolonged LHRH-A treatment prevented pulsatile LH and T secretion compared with control bulls. Mean serum LH did not differ from that of controls on d 4, 8 and 14 of LHRH-A treatment, while serum T was elevated (P less than .01) during the same time periods. Oscillating patterns and mean concentrations of serum FSH were not different between control and LHRH-A-treated bulls. Fifteen days of LHRH-A treatment depressed pituitary LHRH receptor numbers (P less than .05) and pituitary LH (P less than .01) and FSH (P less than .05) concentrations. Testicular LH receptor numbers were elevated (P less than .01), but testicular FSH receptor numbers were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle stimulating hormone binding inhibitor produced by the bacteria Serratia interacts with receptor for follicle stimulating hormone in calf testis membranes.

Bacteria of the genus Serratia, including a strain of Serratia liquifaciens isolated from contaminated porcine follicular fluid, produced an inhibitor of 125I-human follicle-stimulating hormone (hFSH) binding to calf testis membranes in vitro. In order to evaluate its potential usefulness and significance, we undertook studies to identify the site of action of this inhibitor. Large quantities (grams) of inhibitor-containing material (SL-1) were obtained by enrichment culture techniques and its chemical composition was determined. Follicle-stimulating hormone-binding inhibitory activity (FSH-BI) in SL-1 was associated with a protein-containing substance of approximately 30,000 Mr and also with larger Mr (greater than 300,000) forms. Preincubation studies demonstrated that binding inhibition by SL-1 was due to effects on the membranes rather than effects on the radioligand (125I-hFSH). Kinetics studies indicated that FSH inhibition by SL-1 was a relatively slow process that reached steady-state conditions between 23 and 25 h at 20 degrees C, in contrast to FSH, which reached steady-state conditions by 12 h at 20 degrees C. Estimates of FSH-BI activity, e.g., mass required to produce a 50% inhibition of 125I-hFSH binding, varied drastically when these kinetics differences were not taken into account. Our observations emphasize the need to establish steady-state conditions for each ligand before assessing mechanisms of action using Michaelis-Menton assumptions (e.g., competitive binding assays, Scatchard analyses).(ABSTRACT TRUNCATED AT 250 WORDS)