Luteinizing hormone in the hypothalamus of the sheep : its characteristics and binding to receptors

Abstract

Human chorionic gonadotropin (hCG) (5µg) which was iodinated with ImCi Na¹²⁵ I was about 50% and 40% as potent as that which was iodinated with .25 mCi Na¹²⁵ I in radioligand receptor assay (RRA) and in vitro bioassays respectively. The amount of iodine incorporated to the hormone rather than the radioactivity was responsible for these potency losses of the radioiodinated hormone. Preincubation or incubation of porcine granulosa cells in the aqueous extract of sheep hypothalamus resulted in a decrease in the binding of ¹²⁵ I -hCG to these cells. Addition of aprotinin I (.06 - 1.5 TIU/ml) or bacitracin (2 mM) to the extract did not prevent this decrease in binding. The results indicate that the binding decrease was due to receptor occupancy by hypothalamic LH, and was not due to damage to receptors. The ability of ¹²⁵ I -hCG to displace hypothalamic LH from its receptors was dependent on the duration and conditions of the preincubation. The aqueous extract of sheep hypothalamus contained LH which behaved similarly to that of pituitary and skeletal muscle extracts in gel chromatography, RIA, RRA and in vitro bioassays. Hypothalamus including median eminence, hypothalamus minus median eminence and pituitary contained 27µg, 137ng and 1342µg respectively of immunoactive LH per gram wet weight of tissue. Basing on both RRA and in vitro bioassays, hypothalamic LH averaged about 1.5 and .5 as potent as pituitary and skeletal muscle LH respectively. Particulate fractions (1000 x g, 10000 x g and 70000 x g) of sheep hypothalamus were incapable of binding to ¹²⁵ I – oLH or ¹²⁵ I -hCG. This was thought to be due to complete occupancy of hypothalamic LH/hCG receptors by hypothalamic LH. Four techniques for dissociating LH/hCG from its receptors were tested, viz. incubation of LH/hCG - receptor complexes in 1) pH 2-3 buffer, 2) pH 7.4 buffer, 3) buffer containing an excess of free LH/hCG receptors, 4) MgCl₂, 2M buffer. The receptors uncovered by technique 4 retained full binding affinity and capacity. However, using this technique as well as the others no population of specific receptors for LH/hCG could be demonstrated in any hypothalamic fractions.