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Results of studies of the inhibition of human chorionic gonadotropin (HCG)-induced ovarian and uterine weight augmentation in the immature rat by analogs of GnRH are presented. HCG augmented weight gain has been used as a bioassay for measurement of follicle stimulating hormone (FSH). 21-day-old HCG-primed (50 IU twice daily) animals, either intact or hypophysectomized, were injected with GnRH analogs (D-Leu(6), des-GlyNH(2), Pro-ethylamide(9))-GnRH (I). Both the intact and hypophysectomized rat displayed reduced ovarian weight after injection with .2-5 mcg I from Days 22 to 24 (p less than .01). This inhibitory effect was present even when exogenous luteinizing hormone was given in addition to HCG or when FSH was augmented by HCG. Both HCG and HCG + I injected on Days 22-24, significantly advanced the day of vaginal opening (p less than .01). However, when treatment was continued into puberty, vaginal opening was delayed.

The ovulation-inducing and gonadotropin-releasing activities of [d-Leu-6, des-Gly NH2-10, Pro-ethylamide-9]-GnRH (II), were evaluated in rats, rabbits, and sheep. A sc dose of 3.4 ng/100 g body wt of the analog was equal to 160 ng/100 g body wt of GnRH in causing ovulation in the diestrous rat. At these dose levels, the integrated LH release was 1.9 times greater for the analog. Both the time of increase and maximum serum concentrations of LH were delayed after injection of the analog. Oral administration of II and GnRH to the proestrous rat resulted in an ED50 for ovulation of 0.92 and 54 mug/100 g body wt,respectively. Serum levels of LH and FSH were highly variable for the various treatment groups when both releasing substances were administered orally. The intense ovulating activity of II was also evident in the estrous rabbit as indicated by an activity 31 times greater than that of GnRH. Additionally, the analog was at least 50 times more active than GnRH in releasing LH in both the mid-luteal and anestrous ewe. From our experiments with the cycling rat it appears that the intense ovulation-inducing activity of II can be accounted for by the intrinsic LH-releasing activity of the nonapeptide, rather than by a prolonged release stimulus.

Crude extracts of commercially availa-ble ovine, bovine and porcine pineal glands were found to contain high levels of gonadotropin-releasing activity. The gonadotropin-releasing hor-mone (Gn-RH) radioimmunoassay values (porcine, 185 ng/gland; bovine, 500 ng/gland; and ovine, 490 ng/gland) were consistent with values obtained by in vitro Gn-RH bioassay. The pineal glands con-tained 4–10 times as much gonadotropin-releasing activity as that reported in hypothalami of the corresponding species. To verify this high ratio, fresh pineal glands and hypothalami were obtained from 4 ewes and similarly extracted. The pineal:hypothalamus activity ratio was found to be more than 20:1. The bovine and ovine pineal extracts were also tested for the induction of ovulation in the estrous rabbit. Ovulation occurred in all animals treated with the extract at a dose equivalent to 2μg Gn-RH (based on radioimmuno and bioassay results). One of the extracts was further characterized by gel filtration on Sephadex G-25 using 3H-p...

A series of peptide analogs of luteinizing hormone releasing hormone (LH-RH), altered at position 6 and 10, was synthesized and evaluated in vivo for the ability to induce ovulation in the diestrous rat and in vitro for ability to release pituitary luteinizing hormone and follicle stimulating hormone. All the analogs with D-amino acid substitutions at position 6, even those with large bulky side chain, exhibited an amazingly high potency compared with the parent hormone, LH-RH. On the basis of the biological activities, structure-activity relationships in the central part of this molecule were discussed in detail.

The ovulating and gonadotropinreleasing activity of pGlu-His-Trp-Ser-Tyr-Gly- Leu-Arg-Pro-Gly-NH2 (Gn-RH) and the highly potent analog [des-Gly-NH210, Pro-ethylamide9] Gn-RH (I) were compared in the pentobarbitalblocked proestrous rat. Serum LH and FSH were measured by radioimmunoassay and ovulation by the number of ova present in the fallopian tubes. Serum LH at 45 min after injection increased linearly as the dose level of Gn-RH and I increased. Invariably, the response was greater to a given dose of I, than to Gn-RH. Moreover, at the dose levels of Gn-RH, peak serum LH occurred between 15 and 30 min post-treatment, whereas maximal concentrations of LH in the serum occurred 45 min after the injection of I. The greater responses of the serum concentrations of FSH to I than to identical doses of Gn-RH also supports the greater activity of I, although ovulation occurred at dose levels of both Gn-RH and I which did not effect a serum increase. A dose level of 0.051 M£ I induced ovulation in all of the treat...

SummaryThe synthetic decapeptide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Gn-RH) was evaluated for induction of ovulation in the cycling rat. All injections of the decapeptide were made at 9:00 am. At the intravenous dose of 5 μg, Gn-RH induced ovulation only during proestrus in both the normal cycling and pentobarbital-blocked rat. Serum LH elevation following Gn-RH was greater at proestrus than at metestrus or diestrus-1, and least at diestrus-2. Peak serum levels of LH were, respectively 60.6, 33.5, 33.5, and 18.8 ng/ml NIH-LHS16. In the ovulating rat, serum LH elevations after Gn-RH were similar in magnitude and duration to the normal proestrous levels.The authors wish to thank Dr. G. D. Niswender for his generous gift of LH antisera and Jacquelyn Dausman for her excellent technical help.

Five new analogs of luteinizing hormone releasing hormone (LH-RH), des-Gly10-[Ala6]-LH-RH-ethylamide, des-Gly10-[D-Ala6]-LH-RH-ethylamide, des-Gly10-[α-aminoisobutyric acid6]-LH-RH-ethylamide, des-Gly10-[Phe5, D-Ala6]-LH-RH-ethylamide and des-Gly10-[Ile5, D-Ala6]-LH-RH-ethylamide were synthesized and evaluated for the ovulation-inducing activity in the rat, and it was found that the analogs, des-Gly10-[D-Ala6]-LH-RH-ethylamide and des-Gly10-[Phe5, D-Ala6]-LH-RH-ethylamide, were 50 times or more active than the original molecule.

The ovulation-inducing and gonadotropin-releasing activities of [d-Leu-6, des-Gly NH2-10, Pro-ethylamide-9]-GnRH (II), were evaluated in rats, rabbits, and sheep. A sc dose of 3.4 ng/100 g body wt of the analog was equal to 160 ng/100 g body wt of GnRH in causing ovulation in the diestrous rat. At these dose levels, the integrated LH release was 1.9 times greater for the analog. Both the time of increase and maximum serum concentrations of LH were delayed after injection of the analog. Oral administration of II and GnRH to the proestrous rat resulted in an ED50 for ovulation of 0.92 and 54 mug/100 g body wt,respectively. Serum levels of LH and FSH were highly variable for the various treatment groups when both releasing substances were administered orally. The intense ovulating activity of II was also evident in the estrous rabbit as indicated by an activity 31 times greater than that of GnRH. Additionally, the analog was at least 50 times more active than GnRH in releasing LH in both the mid-luteal and anestrous ewe. From our experiments with the cycling rat it appears that the intense ovulation-inducing activity of II can be accounted for by the intrinsic LH-releasing activity of the nonapeptide, rather than by a prolonged release stimulus.Ovulation and gonadotropin-releasing activity of D-Leu6, des-Gly NH2 10, Pro-ethylamide 9 -GnRH (38715) (2) were investigated in rats, rabbits and sheep. When given to the diestrous or proestrous rat, 2 was 47 and 59 times, respectively, more effective than pGlu-His-Trp-Ser-Tyr-Gly -Leu-Arg-Pro-Gly-NG2 (GnRH) in causing ovulation. The analog increased the number of ova shed at the higher dose levels in the diestrous rat (p less than .05) but neither peptide affected the number of ova recovered from proestrus rats. Generally, serum luteinizing hormone and follicle stimulating levels were related to the dose of the peptide. The integrated luteinizing hormone (LH) release was 1.9 times greater for the analog and the time of increase and maximum serum concentrations of LH were delayed after injection of the analog. The analog was at least 50 times more active than GnRH in releasing LH in both the midluteal and anestrous ewe. It appears that the intense ovulation-inducing activity of 2 can be accounted for by the intrinsic LH-releasing activity of the nonapeptide rather than by a prolonged release stimulus.

Five polyamine substances already known to occur naturally in mammalian tissues have been isolated from porcine hypothalamic extracts. Taken together, these substances account for most of the FSH-depleting activity of the crude extracts. Of the polyamines isolated, histamine is present in the highest amount. However, in the assay method used (decrease of pituitary FSH levels in intact and in castrated testosterone-treated male rats) histamine is active only at μg levels; whereas putrescine, which occurs in smaller amounts, is active down to levels of a few ng. Spermidine, spermine and lysine, which have also been identified, are active at intermediate dose levels. The exact roles of the 5 amines in the turnover of pituitary FSH remain to be elucidated. (Endocrinology 82: 742, 1968)

Twenty analogs of luteinizing hormone releasing hormone (LH-RH or pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), i.e., , , [Pro1]-, [Phe2]-, [3-Me-His2]-, [Lys2]-, [Arg2]-, [Ala4]-, [Thr4]-, [Gln5]-, [Cl-Tyr5]-, [di-Cl-Tyr7]-, [Gly7]-, [Ala7]-, [Val7] [Ile7]-, [Nle7]-, [Lys8]-, [Orn8]-, and [Ala10]-LH-RH, were synthesized by the fragment condensation method and some biological properties of these decapeptide amides were studied. On the basis of in , vitro and in , vivo biological activities of these analogs, structure-activity relationships were discussed.

Five new analogs of LH-RH(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), ( Des-Gly-NH 2 10 , Pro-methylamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-propylamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-ethanolamide9)-LH-RH, ( Des-Gly-NH 2 10 , Pro-pyrrolidineamide9)-LH-RH and ( Des-Gly-NH 2 10 , Pro-morpholineamide9)-LH-RH were synthesized and evaluated for the hormonal activity. The activities of these new analogs were compared with those of LH-RH and our previously reported highly potent analog, ( Des-Gly-NH 2 10 , Pro-ethylamide9)-LH-RH. The results demonstrate a significant contribution of the total chain-length of the hormone for the hormonal action.

The decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, corresponding to the amino acid sequence of porcine gonadotropin-releasing hormone (Gn-RH), was synthesized by the solid-phase method. The protected product was purified by chromatography on two silica-gel columns and crystallization from hot methanol. Following hydrogen fluoride deblocking and anion-exchange, the peptide was purified by gel-filtration through Sephadex G-25 followed by Sephadex G-15. This product was analyzed by optical rotation, elemental analysis, thin-layer electrophoresis chromatography, amino acid analysis, and digestion by α-chymotrypsin, leucine amino-peptidase, and carboxypeptidase-A. By these criteria the synthetic material was determined to be optically and chemically, greater than 96% pure. In vitro releasing activity of the peptide was determined by incubation of hemisected anterior pituitaries from male rats in a complete tissue culture media. FSH release was measured by specific bioassay, as well as by radi...