Binding capacity of luteinizing hormone-releasing hormone and its analogues for pituitary receptor sites.

Competition for luteinizing hormone-releasing hormone (LH-RH) receptor sites by the inhibitory analog [D-Phe², D-Trp³, D-Phe⁶]-LH-RH and by the superactive stimulatory analog [D-Trp⁶]-LH-RH was observed in adenohypophysial homogenates incubated at 4°C. Competition for LH-RH binding sites was less evident with adenohypophysial plasma membranes. The binding affinities of these analogues to LH-RH pituitary receptors can explain at least in part their respective action in blocking ovulation and in inducing a greater release of luteinizing hormone and follicle stimulating hormone than the parent hormone.     

Luteinizing hormone-releasing hormone analogs with increased anti-ovulatory activity

A series of LH-RH antagonist analogs has been developed in which inhibitory activities have been increased to a potentially clinically useful level. The new peptides, which are typified by [N-acetyl-D-p-Cl-Phe^1,2, D-Trp³, D-Phe⁶,D-Ala¹⁰]-LH-RH and [N-acetyl-D-Trp^1,3,D-p-Cl-Phe²,D-Phe⁶, D-Ala¹⁰]-LH-RH, most importantly contain new modification to positions 1, 2 and 10, and induce full blockade of ovulation at single doses as low as 10 μg per rat (50 μg/kg). Various ring substituents on D-Trp or D-Phe in position 1 or other D-amino acid replacements in position 10 did not significantly improve anti-ovulatory activity. Incorporation of N-Me-Leu in position 7 was slightly detrimental to activity.     

D-pGlu1,D-Phe2,D-Trp3,6]-LRF. A potent luteinizing hormone releasing factor antagonist in vitro and inhibitor of ovulation in the rat.

The decapeptide D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂ was synthesized in gram quantities by solid phase techniques on a methyl benzhydrylamine resin. This peptide was purified by ion exchange and partition chromatographies. It was fully characterized by amino acid analysis, tlc in four systems, optical rotation and high pressure liquid chromatography (reversed phase) using a triethyl ammonium phosphate buffer. This peptide was found to inhibit ovulation by 100% in the rat at a subcutaneous dose of 250μg in corn oil when administered at noon on the day of proestrus. Furthermore, it was found to be long acting since 1.5mg blocked ovulation in 9 out of 10 rats when administered as early as 19 hrs before the afternoon of proestrus. [D-pGlu¹, D-Phe², D-Trp^3,6]-LRF reduces the amount of LH normally secreted in response to LRF by 50% at a molar ratio (IDR₅₀) of 3/1 ([antagonist])/[LRF]). Three other analogs, equally well-characterized and with the D-pGlu¹ modification, i.e. [D-pGlu¹, D-Phe², Pro³, D-Trp⁶]-LRF, [D-pGlu¹, D-Phe², Phe³, D-Trp⁶]-LRF and [D-pGlu¹, D-Phe^2,6, D-Trp³]-LRF were found to be somewhat less potent ($\text{IDR}{}_{50}\text{=}\text{150}\text{1}\text{,}\text{60}\text{1}\text{,}\text{5}\text{1}$) respectively.     

Reduction of LH-RH pituitary and estradiol uterine binding sites by a superactive analog of luteinizing hormone-releasing hormone

The ability of the Luteinizing Hormone-Releasing Hormone (LH-RH) analogs to displace LH-RH from its pituitary receptors was evaluated $\text{in}\text{vitro}$. The two superactive analogs tested showed higher potency than the antagonists and LH-RH itself, D-Trp⁶-LH-RH being the most potent. The LH-RH specific binding activity in the pituitary fluctuated throughout the age of the rats. The highest number of LH-RH binding sites were seen on day 35 of age (276 fmol × 10^?2/pit) and an increment was induced by 0.05 μg D-Trp⁶-LH-RH (400 fmol × 10^?2/pit). However, 1 μg D-Trp⁶-LH-RH reduced the binding of LH-RH at all the times studied. In the control animals the number of estradiol binding sites increased on day 42 of age, and 0.05 μg D-Trp⁶-LH-RH augmented them on day 35 of age. On the contrary, 1 μg D-Trp⁶-LH-RH diminished the estradiol uterine receptors at all the times studied. Similar results were obtained in the ovariectomized-hypophysectomized rats on day 35 of age. Our studies demonstrated a biphasic action of D-Trp⁶-LH-RH on LH-RH pituitary receptors and a direct effect on uterus which could be mediated through the uterine estradiol receptors.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Synthesis and biological activities of pseudopeptide analogues of LH-RH: agonists and antagonists

Using solid phase methods, seven agonist and antagonist analogues of LH-RH have been prepared containing enzyme-resistant CH₂S linkages as selected amide bond replacements. Agonists modified at the 5–6, 6–7 and 9–10 position had 2, < 0.1, and 10% of the $\text{in}\text{vitro}$ activity of LH-RH, respectively. Among potential antagonists, 6–7 position analogues showed only minimal inhibitory activity but N- and C-terminal modified analogues retained substantial LH-RH-LH and FSH inhibitory activity. In addition, a 1–2 position methylene thioether analogue of the parent [Ac-Pro¹, D-Phe², D-Trp^3,6]LH-RH antagonist was completely inhibitory at 30 ng $\text{in}\text{vitro}$ and represents the first such structure-modification that may be at least as active as its corresponding amide linked congener. However, neither 1–2 nor 9–10 methylene thioether position antagonists showed $\text{in}\text{vivo}$ antiovulatory activity at the 250 μg level.     

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10.

Potent agonist and antagonist analogues of luliberin containing an azaglycine residue in position 10 were synthesised and tested in androgen-sterilised constant-oestrus rats. The agonist, [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin, induced ovulation at a dose of 6ng/rat i.v., 10μg/rat p.o. and was at least five times as potent as [D-Ser(Bu^t)⁶, des-Gly-NH₂¹⁰, Pro-ethylamide⁹]-luliberin. [D-Ser(Bu^t)⁶, Azgly¹⁰]-luliberin (1μg/rat) also prevented HCG-induced increases in ovarian and uterine weight in immature rats and was a highly potent antitumour agent when given to rats bearing DMBA-induced mammary tumours. The antagonist, [D-Phe², D-Phe⁶, Azgly¹⁰]-luliberin at a dose of 15μg/rat completely inhibited ovulation induced by luliberin (0.5μg/rat), whereas [D-Phe², D-Phe⁶]-luliberin lost activity below 125μg/rat.     

Reduction of testicular luteinizing hormone/human chorionic gonadotropin receptors by [D-Trp6]-luteinizing hormone releasing hormone in hypophysectomized rats.

Adult and immature male rats were hypophysectomized and injected daily with saline or 0.2 or 2 μg of superactive Luteinizing Hormone Releasing Hormone (LHRH) agonist, [D-Trp⁶]-LHRH subcutaneously for seven days - with, or without, concomitant treatment of 1 IU Human Chorionic Gonadotropin (hCG) or 50 IU Pregnant Mare Serum. The administration of [D-Trp⁶]-LHRH reduced Luteinizing Hormone/Human Chorionic Gonadotropin receptors in all cases. The magnitude of this reduction was dose-related. As small a dose as 0.2 μg of the peptide resulted in approximately a 72% reduction of the receptors. The results suggest a direct action of [D-Trp⁶]-LHRH on the testis. It also indicated that reduction of testicular Luteinizing Hormone/Human Chorionic Gonadotropin receptors by the peptide is not necessarily due to the over-stimulation of Luteinizing Hormone (LH) release from the pituitary through a “down regulation” mechanism.     

Resistance of the mouse to the antifertility effects of LHRH agonists

In the mouse, the LH-releasing activity of the LHRH agonist, D-Trp⁶-N^α-Meleu⁷-DesGly¹⁰-Pro⁹-NHEt-LHRH (Wy-40, 972), was established by its ability both to induce ovulation when administered at 1600 hours on the second day of diestrus and to elevate serum LH in adult males. While Wy-40, 972 was only slightly less active in terms of these end points than it was in the rat, the predictive and possibly causal association between LH-releasing and antifertility activity established for this LHRH analog in the rat could not be clearly identified in the mouse. A total daily dose of 1000 μg Wy-40, 972/mouse was required to completely inhibit pregnancy during days 1–7 of pregnancy and produced only partial inhibition during days 7–12. This dose represents, on a body weight basis, 8250 times the 100 percent effective pregnancy-terminating dose for the rat during equivalent intervals. The resistance of the mouse to the antifertility activity of Wy-40, 972 was found not to be restricted to this particular LHRH analog or to the reproductive state. Administration of another potent LHRH analog, D-Ala⁶-DesGly¹⁰-Pro⁹-NHEt-LHRH (Wy-18,481), to adult male mice at a dose of 100 μg/mouse/day for up to 14 days had no inhibitory effect on the weights of the testes or sex accesory organs. This dose of Wy-18,481 is 7500 times that necessary for significant reduction of these reproductive organ weights in rats within 7 days of treatment. Investigations as to the nature of the mouse's apparently divergent response to the LHRH agonists may further elucidate the antifertility mechanism of such compounds in susceptible species.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Effect of somatostatin and two of its analogues on basal and arginine-stimulated insulin and glucagon secretion in the dog

Two analogues of somatostatin (d-Trp⁸,d-Cys¹⁴-somatostatin, and the octapeptide DesAA^{1,2,3,4,13,14},d-Trp⁸,GABA¹²-somatostatin) were compared with somatostatin using infusions, of 0.1 and 0.5 μg · kg^?1 · min^?1 in conscious dogs. Basal concentrations of insulin and glucagon were markedly and similarly lowered by all three somatostatin (SRIF) compounds at either dose. Arginine stimulation of insulin and glucagon secretion was entirely abolished by SRIF and by the octapeptide during infusion at 0.1 μg · kg^?1 · min^?1 but both hormones were only partly inhibited by d-Trp⁸,d-Cys¹⁴-SRIF. The higher dose (0.5 μg · kg^?1 · min^?1) of all three SRIF peptides lowered plasma insulin and glucagon before and during arginine stimulation. The recovery of plasma insulin and glucagon was delayed after discontinuation of the d-Trp⁸,d-Cys¹⁴-SRIF, and particularly after the octapeptide when compared with SRIF suggesting a longer duration of action of the analogues.The results did not confirm the previously suggested selective suppression of glucagon by d-Trp⁸,d-Cys¹⁴-SRIF. The new octapeptide appears to be promising for future clinical studies due to its potent inhibitory effect on insulin and glucagon, and its prolonged duration of action.     

Immunoreactive-beta-endorphin, -adrenocorticotropin and -calcitonin in extracts of anaplastic or differentiated (rat) medullary thyroid carcinoma.

Fifteen analogs of luliberin (2, LRH) were synthesized by the solid phase method and examined for their ability to block ovulation in the rat. Two analogs, [Ac-DAla¹,DPhe²,DTrp^3,6]-LRH and [Ac-DPhe¹,DPhe²,DTrp^3,6]-LRH, each blocked ovulation at a single injection dose of 250 μg administered at noon on the day of proestrus; three peptides, [Ac-DPro¹,DPhe²,DTrp^3,6]-LRH, [Ac-DThi¹,DPhe²,DTrp^3,6]-LRH and [Ac-DTrp¹,DPhe²,DTrp^3,6]-LRH, were effective at doses of 500 μg each; and four others, [Ac-DTrp¹,DPhe²,DTrp³,DTrp(Nps)⁶]-LRH, [Chlorambucil-DPhe¹, DPhe², DTrp^3,6]-LRH, [1,DThi²,DTrp^3,6]-LRH and [(2-DLys⁶]-LRH, gave partial inhibition at doses tested.     

Induced ovulation in cultured tawny puffer Takifugu flavidus by injections of LHRH‐a

To determine the effective LHRH‐a dosage for cultured tawny puffer ovulation, the effects of 10, 30, 50, and 70 μg kg^?1 body weight (BW) LHRH‐a were tested with two injections in 2009, and by a single injection (5, 10, 30, 50, and 70 μg kg^?1 BW) in 2010. Ovulation success and egg quality were assessed. All females spawned after being treated with 10 to 70 μg kg^?1 BW LHRH‐a in 2009 and 2010. Ovulation success was only 20.0% with the 5 μg kg^?1 BW treatment, significantly lower (P < 0.05) than in the other treatment groups. No females injected with physiologic saline spawned in the control groups in 2009 and 2010. There was no significant difference (P > 0.05) among the 10, 30, 50, and 70 μg kg^?1 BW treatment groups in terms of ovulation success, ovulation time, egg production, fertilization rate, hatch rate, and viability of 24 h post‐hatch larvae in 2009 and 2010. None of the treatments caused any mortality among the females during the experimental periods. Results suggest that the treatment using only LHRH‐a effectively and reliably induces ovulation in cultured tawny puffer broodstocks, whether through a single injection or through two injections. Effective and most economical dosage of LHRH‐a is 10 μg kg^?1 BW. Determination of the effective LHRH‐a dosage is an important step in the development of tawny puffer fish culture.     

Resistance to enzymic degradation of LH-RH analogues possessing increased biological activity.

Three analogues of LH-RH in which Dextrarotatory amino acids were substituted for the Gly⁶, and two additional analogues in which the Leu⁷ residue was also modified, were subjected to enzymic preparations derived from rat hypothalamus or anterior pituitary. These enzymes, known to cleave LH-RH, preferentially at the Gly⁶-Leu⁷ position, proved less effective in degrading all the analogues tested. Among the Gly⁶ substituted analogues, [D-Trp⁶] LH-RH, having the highest LH-releasing activity, was most resistant to degradation. Additional modification, at position 7, although rendering the analogues immune to enzymic attack, did not further enhance their biological potency. These data suggest that degradation of LH-RH is a physiological determinant of its biological activity and has therefore to be considered with on designing new, potent analogues of the hormone.     

Antiovulatory antagonists of LHRH related to antide

We report 104 analogues of the potent antiovulatory antagonist of LHRH, N-Ac-D -Nal-D -Cpa-D -Pal-Ser-Lys(Nic)-D -Lys(Nic)-Leu-Ilys-Pro-D -Ala-NH₂, Antide. We replaced the Nic group in Antide with other acyl substituents to modulate size, hydrophilicity or basicity of the molecule, we also replaced th Lys residues with shorter basic amino acids, and made cyclic 5/6 analogues as well as position 5 or 6 dimers. We substituted Ilys⁸ with other alkyl groups and acyl derivatives. When injected in 0.1% DMSO in water in a typical antiovulatory (AO) assay, Antide gives six rats ovulating out of eight (6/8) at 2 μg, 4/8 at 4 μg, and the histamine release assay (HRA), ED₅₀ is >300 μg/ml; [Lys(N-Isobutyl)⁸]Antide gave 2/8 at 2 μg/rat; [Lys (8-Qis)⁵]Antide gave 1/8 at 1 μg, and 0/8 at 2 μg, and in the HRA ED₅₀, 22 μg/ml; [D -Lys(8-Qis)⁶]Antide gave 4/8 at 1 μg and 0/8 at 2 μg, and in the HRA, ED₅₀ was 27 μg/ml; [Lys(8-Qic)⁸] gave 5/8 at 1 μg, 1/8 at 2 μg/ [Lys(2-Pyc)⁵]Antide gave 5/8 at 1 μg and 0/8 at 2 μg, and in the HRA ED₅₀ was 116 μg/ml; [D -Lys (2-Pyc)⁶]Antide gave 3/8 at 1 μg, and in the HRA, ED₅₀ was 100 - >300 μg/ml; [Lys(2-Pyc)⁵,D -Lys(2-Pyc)⁶]Antide gave 2/8 at 1 μg. The substitutions of the Nic groups of Antide at Lys⁵ or D -Lys⁶ with 8-Qis or with 2-Pyc groups seem to give highly potent antiovulatory antagonists of LHRH and constitute significant new leads to generate potent antiovulatory compounds endowed with moderate or low histamine release.     

A new category of ovulation inhibitors: linear LH-RH analogues having more than ten residues.

A linear analogue of the luteinizing hormone-releasing hormone, longer than a decapeptide, is described for the first time, which is equivalent in potency to the best known inhibitors of ovulation, and which constitutes an important new lead to the design of inhibitors of even greater potency. At a dosage of 200 μg/rat, the undecapeptide [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH caused 100% inhibition of ovulation. The related analogues, [(1, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH and [(Gly-Pro)¹, $\text{D}$-Phe², $\text{D}$-Trp³, $\text{D}$-Trp⁶]-LH-RH, were less active, $\text{in}\text{vivo}$. All of these undecapeptides inhibited the action of 0.6 ng/ml of LH-RH by greater than 50% at the very low level of 10 ng/ml.     

Effect of intermittent injections of gonadotrophin releasing hormone at various frequencies on the release of luteinizing hormone and ovulation in dairy cows

Gonadotrophin releasing hormone (GnRH, 5 μg every 4 h) was administered to six dairy cows between days 5 and 10 post-partum and the release of luteinizing hormone (LH) and the onset of ovulation were determined. LH was measured using a specific radioimmunoassay and the occurrence of ovulation was assessed from changes in the concentration of progesterone in milk. Treatment with GnRH resulted in a median time of first ovulation of 17.0 days after calving. This was less (P < 0.05) than that observed for control cows (21.5 days, n = 7). Determinations of plasma LH concentrations over an 8-h period on days 6 and 10 post-partum indicated that there was a tendency for GnRH-treated cows to have higher levels of LH on these days. The 5 μg dose of GnRH did not repeatably induce a release of LH between days 6 and 10. Endogenous pulsatile release of LH did, however, increase in frequency from 3.18 pulses per 8 h on day 6 to 5.18 pulses per 8 h on day 14 post-partum (P < 0.01).In a second experiment groups of 20 cows were treated with either 5 μg GnRH every 4 h or 15 μg GnRH every 12 h from days 5 to 10 post-partum. Seventeen untreated cows served as controls. The median times to first ovulation were 27.0 days for the control cows, 22.5 days for those cows treated with 5 μg GnRH every 4 h and 17.0 days for cows treated with 15 μg every 12 h. The latter treatment significantly advanced the time of first ovulation (P < 0.05) relative to controls. This difference had, however, disappeared by the time of the second and third ovulations. Primiparous cows ovulated later (P < 0.01) than the pluriparous cows in the group treated with 5 μg GnRH every 4 h. This was a major reason for the lack of effect of this treatment. Some treated cows were blood sampled at frequent intervals on day 8 to evaluate the LH responses to GnRH injections. The administration of 5 μg GnRH on day 8 did not elicit a pulse of LH which could be distinguished from endogenous pulsatile secretion at this time. The dose of 15 μg on this day did, however, elicit a more defined pulse on some, but not all, occasions.The injection of a small dose of GnRH twice a day from day 5 to day 10 after calving, therefore, advanced the time of first ovulation in dairy cows by 10 days.     

Substance P-related antagonists inhibit vasopressin and bombesin but not 5′-3-O-(thio) triphosphate-stimulated inositol phosphate production in swiss 3T3 cells

The substance P (SP) analogues [DArg¹, DPhe⁵, DTrp^7,9, Leu¹¹] SP (AntD) and [Arg⁶, DTrp^7,9, MePhe⁸] SP (6–11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5′-3-O-(thio) triphosphate (GTPγS) stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC₅₀ of 0.75 μM and 2 μM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC₅₀ of 1 μM. Strikingly, neither AntD up to 10 μM nor AntG up to 20 μM was able to inhibit GTPγS-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 μM AntD or 20 μM AntG. However, neither antagonist affected the dose response of GTPγS-stimulated inositol phosphate generation. Furthermore, 20 μM AntD had no effect on AIF^?₄-induced inositol phosphates in COS-1 cells transfected with G_αq. AntD inhibited [³H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [³H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca²⁺ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production. © 1995 Wiley-Liss, Inc.     

Analgesic effect of antagonists of substance P

[D-Pro²,D-Phe⁷,D-Trp⁹]-SP and [D-Pro²,D-Trp^7,9]-SP havebeen shown to be antagonists of substance P. The hindlimb scratching syndrome of mice, known to be caused by substance P was absent when these peptides were injected into substance P-treated mice. Substance P shortens “tail withdrawal time” from hot water; the two peptides greatly prolonged tail withdrawal time. Antidromic stimulation of the saphenous nerve (rat), known to release substance P and to induce vasodilatation plasma extravasation, was also greatly inhibited by [D-Pro²,D-Phe⁷,D-Trp⁹]-SP. These peptides presumably cause anti-nociceptor effects (analgesia) by inhibition of substance P at receptors and favor the concept that substance P is a sensory neurotransmitter of nociceptive messages.     

Effect of prolonged administration of small doses of LH-RH and LH-RH analogue [D-Ser (But)6] Des Gly-NH210 ethylamide on the release of LH and induction of ovulation in mid-anoestrous ewes

A natural process of LH release and induction of ovulation in anoestrous ewes was simulated by prolonged administration of small doses of LH-RH and its analogue [D-Ser(But)⁶] Des Gly-NH₂¹⁰ ethylamide. In the first series of experiments on 40 Merino ewes infusions of LH-RH were made into the maxillaris interna artery for 6 consecutive days for 6 h each day. Total doses of 24.0, 26.0, 28.0 and 32.0 μg per animal of varying and progressively increasing daily quantities of the hormone were administered in groups I, II, III and IV, respectively. In group V the animals were infused with a total dose of 28.0 μg LH-RH and injected additionally i.m. with 3.0 μg 17β-oestradiol on days 4 and 5 of the infusion of LH-RH. Ovulation did not occur earlier than days 4, 5 and 6 after the beginning of infusions. The highest number of positive reactions occurred in group IV ($\text{8}\text{10}$) and in group V ($\text{7}\text{8}$ animals). The pattern of LH peaks in general was correlated with the time of ovulations. The LH concentrations of the preovulatory peaks in experimental ewes were mostly lower than those in naturally ovulating animals. The corpora lutea were functional during the first 7 days after ovulation.In the second series of experiments on 26 Merino ewes the LH-RH analogue [D-Ser -(But)⁶] Des Gly-NH₂¹⁰ ethylamide was injected i.m. or i.a. for 6 consecutive days. Total doses of 15.5, 9.5 and 7.5 μg of the analogue per animal, administered at varying and progressively increasing daily doses in respective groups, induced several surges of LH in the same individuals for 2 or even 3 consecutive days. Corpora lutea and degenerating follicles in the form of cysts were found in the ovaries of animals of these groups. Very small daily doses ranging from 0.1 μg administered during the first 3 days, to 1.5 μg on day 5 of the treatment, released one surge of LH on day 5 of the treatment in all individuals with peaks ranging from 30.0 to 58.0 ng/ml and induction of ovulation with almost normal luteal function. On the basis of these experiments it is suggested that the evaluation of the effect of active substance (LH-RH or its analogue), its suitability and application of rightly chosen doses to induce the full physiological process of ovulation should be based not only on the release of LH and luteal function but also on tests of the ability of the released ovum to undergo fertilization and its further development.