Gonadotropin-releasing peptide from human follicular fluid: isolation, characterization, and chemical synthesis.

A gonadotropin-releasing peptide has been isolated from human follicular fluid. Its amino acid composition and sequence are completely different from the hypothalamic lutropin-releasing hormone. It is designated human follicular gonadotropin-releasing peptide and abbreviated as hF-GRP. The primary structure of this peptide (H-Thr-Asp-Thr-Ser-His-His-Asp-Gln-Asp-His-Pro-Thr-Phe-Asn-OH) has been confirmed by chemical synthesis. In the mouse pituitary incubation assay, the ED50 value for follitropin or lutropin release is estimated to be 1.2-1.6 nM.

Use of pituitary cells in primary culture to study the regulation of gonadotropin hormone (GtH) secretion in rainbow trout: setting up and validating the system as assessed by its responsiveness to mammalian and salmon gonadotropin releasing hormone.

To study the regulation of gonadotropin secretion in rainbow trout in vitro, a method for preparing primary cultures of dispersed pituitary cells is described. Cells were dispersed by collagenase 0.1% in Hank's saline solution for 20 hr at 12 degrees and a high yield of viable cells was obtained. Attempts to improve cell functioning were made by varying culture conditions (density of cells initially plated, age of the culture). Cell functioning was assessed by their ability to respond to increasing doses of mammalian and salmon GnRH. Pituitaries were collected from spermiating males whose pituitaries are known to be sensitive to mammalian GnRH in vivo. Using 96-well plates, optimal conditions for good biological activity, are initial plating with 6.2 X 10(4) cells, incubation with GnRH for 24 hr on the third day after plating. In these conditions mammalian analog and salmon GnRH induced an increase in GtH release for doses ranging from 10(-9) to 10(-6) M. The GtH released during the GnRH incubation period does not decrease the sensitivity of the system since addition of 20 ng of GtH at the beginning of incubation does not modify the response profile.

Gonadocrinins: peptides in ovarian follicular fluid stimulating the secretion of pituitary gonadotropins.

A newly discovered small peptide purified from rat follicular fluid stimulates the pituitary to release FSH and LH in vitro as well as in vivo. Dialysates of crude acid extracts of ovarian follicular tissue and fluid from rats pretreated with PMS gonadotropin stimulate the secretion of both LH and FSH, but not PRL, GH, or TSH, in a pituitary monolayer culture system. This stimulating factor, named gonadocrinin for operational facility, is smaller than 3500 daltons; its biological activity disappears after treatment with trypsin. Gonadocrinin is not recognized by two-antisera binding the decapeptide LRF even though D-Phe2,D-Trp6-LR, an LRF analog antagonist, competitively inhibits the activity of ovarian gonadocrinin. Cultured rat granulosa cells also secret substances with gonadocrinin activity in vitro, indicating that the granulosa cells probably are in vivo the source of gonadocrinin. A crude preparation of gonadocrinin given iv to rats on the second day of diestrus induced secretion of LH comparable to that produced by a 250-ng LRF injection. Gonadocrinin has chemical characteristics different from those of LRF. When purified gonadocrinin or LRF was applied to an identical isocratic high pressure liquid chromatography system, LRF was eluted at a position different from that of gonadocrinin, indicating that, chemically, gonadocrinin is not identical to the hypothalamic decapeptide, LRF.

[Gonadostatin and gonadocrinin, polypeptides of ovarian origin with hypophysiotropic activity]. / Gonadostatine et gonadocrinine, polypeptides d'origine ovarienne à activités hypophysiotropes.

Crude acetic acid extract of Rat ovaries pretreated with pregnant mare serum (PMSG) contains native peptides with two types of separable biological activities: one, molecular weight greater than 10,000 dalton inhibits the secretion of both LH and FSH as stimulated by luteinizing hormone releasing factor (LRF) in a pituitary monolayer culture system and is referred to as gonadostatin; the other, less than 3,500 dalton, stimulates the secretion of gonadotropins and is designated as gonadocrinin. The biological activities of ovarian gonadocrinin can be competitively inhibited by an LRF-analog-antagonist, D-Phe2, D-Trp6-LRF. These ovarian peptides may participate in physiological control of pituitary LH/FSH secretion.

[Hypothalamic peptide hormones]. / Die Peptidhormone des Hypothalamus.

After a review of the significance of the releasing and inhibiting polypeptide hormones of the hypothalamus, the clinical significance of the gonadotropin-releasing-hormone and the thyrotropin releasing hormone, is discussed in detail.

Pineal peptides.

In pineal extracts several activities have been observed which cannot be explained on the basis of their content of melatonin or other pineal indoles to which an antigonadotropic activity is ascribed. The significance of structurally identified and unidentified substances is not fully understood. The list includes a neurohypophysial peptide, vasotocin (AVT), hypothalamic releasing factors and certain unidentified substances including the antigonadotropin under investigation in our laboratory. Our initial experiments demonstrated the presence of a non-melatonin antigonadotropin (PAG) in partially purified extracts of bovine, ovine, rat and human pineals. Purification by ion exchange chromatography and amino acid analyses have shown that this substance is not AVT. Recent purification studies include paper chromatography and high performance chromatography as final steps. It is concluded that if the active principle in a peptide, or contains a peptide moiety important for the biological activity, it may be present only in minute amounts in the puriest fractions derived and the quantities of recoverable material may be similar to those amounts of releasing factors recoverable from hypothalamic tissue. It is anticipated that large scale preparative methods will be required for structural determination. High performance chromatography may prove to be extremely useful in future studies.

A specific radio-immunoassay for gonadotrophin-releasing hormone.

A specific antiserum has been made to synthetic gonadotrophin-releasing hormone (GnRH) conjugated to keyhole limpet haemocyanin and appears to be directed against amino acids 6-8 of this decapeptide. This has allowed the development of a radio-immunoassay for GnRH sensitive to 5 picograms per tube. Although it is easily measurable in hypothalamic extracts, we have failed to detect GnRH in plasma and urine from normal subjects and menopausal women.