Further characterization of the direct inhibitory effect of LHRH agonists at the testicular level in the rat

To further clarify the relative importance of the pituitary and gonadal sites of LHRH action, intact and hypophysectomized adult male rats were treated with hCG for 7 days, in the presence or absence of simultaneous treatment with increasing doses of the LHRH agonist [D-Ser(TBU)6des-Gly-NH2(10)]LHRH ethylamide, Buserelin (0.025, 0.25, 2.5 or 25 micrograms/rat, twice daily). Daily treatment of intact adult rats with hCG (25 IU) markedly increased ventral prostate and seminal vesicle weight, while a dose-dependent inhibition of the effect was observed following combined administration of Buserelin. In hypophysectomized rats, treatment with hCG resulted in a partial restoration of ventral prostate and seminal vesicle weight, while combined treatment with a high dose of the LHRH agonist (25 micrograms, twice daily) partially (P less than 0.05) inhibited the stimulatory effect of hCG. LH/hCG receptors were almost completely inhibited after hCG injection alone and a further decrease was observed in the presence of simultaneous LHRH agonist treatment. The hCG-induced stimulation of GH/PRL receptors was counteracted by Buserelin treatment in hypophysectomized animals. The present data demonstrate that although LHRH-induced LH release has been shown to play a major role in the loss of testicular functions induced by low doses of LHRH agonists in the rat, a direct inhibitory action of LHRH agonists can be exerted at the testicular level at high doses of the peptide.     

The prolonged action of the LHRH agonist buserelin (HOE 766) may be due to prolonged binding to the LHRH receptor

Hemi-pituitary glands of ovariectomized rats were superfused for 4 h with either LHRH or the analog buserelin (HOE 766) at several concentrations, and thereafter with medium only for another 1.5 h. In a further experiment glands were exposed for 2.5 h to LHRH or buserelin at a single concentration (5 ng/ml) and subsequently for another 2.5 h to either the same agonist (LHRH or buserelin) alone (5 ng/ml), the agonist plus an LHRH-antagonist (ORG 30093, 1000 ng/ml), the LHRH- antagonist alone, or medium alone. LHRH and buserelin stimulated gonadotropin release equally well. After cessation of this stimulation, the gonadotropin release by the buserelin-treated pituitary glands and the glands, treated with the highest dose of LHRH (1000 ng/ml), continued, while the release by the glands, treated with the lower doses of LHRH, declined. The LHRH-antagonist completely blocked the release of LH, stimulated by buserelin or LHRH, as well as the prolonged activation of the release, caused by buserelin pre-treatment. In a superfusion experiment with pituitary cell aggregates of 14-day-old intact female rats, buserelin stimulated the release of LH much more effectively than LHRH itself. Moreover, the release caused by buserelin declined more slowly after cessation of the stimulation. Finally, in a pituitary cell monolayer culture the Kd's of LHRH, buserelin and the antagonist were determined as 4.7 X 10(-9) M, 2.4 X 10(-10) M and 4.6 X 10(-9) respectively. It was concluded that the estimates of the potencies of LHRH and buserelin depend on the choice of the test-system. It is suggested that the long duration of action of buserelin is at least partly due to prolonged binding to the LHRH-receptor.     

Relationship between release of LH and incorporation of tritiated thymidine in the anterior pituitary gland of the castrated male rat: effect of LHRH and its highly active analogue buserelin

Castration of the adult male rat significantly (P less than 0.01) increased the concentration of LH in serum and the incorporation of (3H) thymidine into the pituitary DNA. The administration of a single dose of LHRH or its analogue buserelin stimulated the release of LH but it did not modify (3H) thymidine incorporation. When multiple doses of LHRH or buserelin were injected, there was a significant (P less than 0.01) inhibition of LH release and also the incorporation of (3H) thymidine into the DNA of the anterior pituitary gland was significantly (P less than 0.01) diminished. These observations are compatible with the idea of the close relationship between hormonal release and DNA synthesis in the anterior pituitary gland of the rat.     

Inhibitory effects of a luteinizing-hormone-releasing hormone agonist implant on ovine fetal gonadotrophin secretion and pituitary sensitivity to luteinizing-hormone-releasing hormone.

Sheep fetuses at day 70 of gestation (term = 145 days) were implanted subcutaneously with a biodegradable implant containing a luteinizing-hormone-releasing hormone (LHRH) agonist (buserelin) to investigate whether treatment with LHRH agonist would induce a state of desensitization of the fetal gonadotrophs and thus influence fetal gonadal development. Treatment with the LHRH agonist for 35-40 days caused a significant reduction in mean fetal plasma concentrations of LH and follicle-stimulating hormone (FSH) compared with control fetuses. LH pulses were evident in control fetuses but were completely abolished by buserelin treatment. Furthermore, the pituitary content of LH and FSH was significantly depleted in fetuses implanted with LHRH agonist. A bolus intravenous injection of 500 ng LHRH given to control fetuses caused a rapid and significant increase in plasma LH and FSH concentrations which was sustained for at least 60 min after injection. Pretreatment with buserelin completely abolished the LH and FSH responses to a bolus injection of LHRH. There were no differences between the sexes in fetal gonadotrophin concentrations or pituitary sensitivity to LHRH in control or agonist-treated fetuses. Furthermore, buserelin treatment for 35-40 days had no effect on the morphological appearance of the fetal gonads when compared with control fetuses, at least to day 110 of pregnancy. These results provide evidence for the induction of a state of desensitization of the LHRH receptors of the fetal pituitary gonadotrophs following long-term treatment with an LHRH agonist, but provide no evidence for a role for gonadotrophin secretion in gonadal development at this stage in fetal life.     

The rapid effects of luteinizing hormone releasing hormone agonists and antagonists on the mouse pituitary in vitro

The effect of luteinizing hormone releasing hormone (LHRH) and its analogs on the release of FSH and LH by 20 day old whole mouse pituitary incubated in vitro for 3-4 hrs was investigated. Three agonistic analogs (AY 25650, 25205 and Buserelin) all of which are reported to be superactive in vivo showed approximately the same potency in this in vitro test system. Preincubation of the pituitaries for 1 h with the antagonistic analogs [Ac Dp Cl Phe1,2, D Trp3, D Phe6, D Ala10] LHRH and [Ac Dp Cl Phe1,2, D Trp3, D Arg6, D Ala10] LHRH inhibited the secretion of LH and FSH induced by 2.5 x 10(-9)M LHRH. The inhibitory response was dose dependent. The continued presence of the antagonists was not required for effective suppression of the LHRH effect. Experiments designed to find out the minimum time required for eliciting suppression of LHRH revealed that preincubation of the pituitary with the second antagonist for 5 mins followed by removal was adequate to produce effective inhibition of gonadotropin release. At lower doses of the antagonist, LH release was more effectively inhibited than FSH release. The results suggest that antagonistic analogs can effectively bind to LHRH receptors in the whole pituitary incubation preventing the subsequent action of LHRH. With the present incubation system assessment of bioactive LH and FSH release is possible within 24 hrs.     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo. A histological and immunohistochemical study

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapasules of a biodegradable polymer poly(DL-lactide-co-glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [D-Trp6]-LHRH and microcapsules of the LHRH antagonist SB-75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue-compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [D-Trp6]-LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [D-Trp6]-LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.     

The different mechanisms for suppression of pituitary and testicular function

The differential mechanisms reducing androgen secretion by LHRH agonists are discussed with relevance to clinical therapy. LH secretion can be desensitised by exposure to agonists using high doses, frequent injections or sustained release/constant infusion. The desensitized pituitary is refractory to hypothalamic stimulation. Pituitary receptor suppression is associated with depletion of pituitary gonadotrophin content, and a decline of LH and FSH secretion to a basal rate. Recovery of LH responsiveness to endogenous LHRH stimulation requires restitution of gonadotrophin content (about 7 days in rats). After long-term infusions in normal men, testosterone secretion recovers within 7-10 days. The binding capacity of testicular LH/hCG receptors is reduced in rats after supraphysiological gonadotrophin stimulation, by agonists or directly by hCG, concomitantly the steroidogenic capacity of the testis in vitro is impaired. Qualitative changes in androgen biosynthesis are a marked fall in testosterone production and dose-dependent enhancement of progesterone production. After 12 months of buserelin injections, the changes in hCG-stimulated rat testes are an increased ratio of progesterone/17-OH-progesterone (inhibition of 17-hydroxylase), a reduced capacity for secretion of androstenedione and testosterone (block of 17,20-desmolase), and increased 5 alpha-pregnane-3,20-dione (this steroid inhibits the 17,20-desmolase, similarly to progesterone). After treatment, Leydig cell function recovers completely. Leydig cell hyperplasia is observed as a result of the steroidogenic changes. These findings in rats have not been observed in dogs, monkeys or in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of buserelin injection and deslorelin (GnRH-agonist) implants on plasma progesterone, LH, accessory CL formation, follicle and corpus luteum dynamics in Holstein cows

The influence of Buserelin injection and Deslorelin (a GnRH analogue) implants administered on Day 5 of the estrous cycle on plasma concentrations of LH and progesterone (P4), accessory CL formation, and follicle and CL dynamics was examined in nonlactating Holstein cows. On Day 5 (Day 1 = ovulation) following a synchronized estrus, 24 cows were assigned randomly (n = 4 per group) to receive 2 mL saline, i.m. (control), 8 micrograms, i.m. Buserelin or a subcutaneous Deslorelin (DES) implant in concentrations of 75 micrograms, 150 micrograms, 700 micrograms or 2100 micrograms. Blood samples were collected (for LH assay) at 30-min intervals for 2 h before and 12 h after GnRH-treatment from cows assigned to Buserelin, DES-700 micrograms and DES-2100 micrograms treatments and thereafter at 4-h intervals for 48 h. Beginning 24 h after treatment, ovaries were examined by ultrasound at 2-h intervals until ovulation was confirmed. Thereafter, ultrasonography and blood sampling (for P4 assay) was performed daily until a spontaneous ovulation before Day 45. A greater release of LH occurred in response to Deslorelin implants than to Buserelin injection (P < 0.01). Basal levels of LH between 12 and 48 h were higher in DES-700 micrograms group than in DES-2100 micrograms and Buserelin (P < 0.05). The first wave dominant follicle ovulated in all cows following GnRH treatment. Days to CL regression did not differ between treatments, but return to estrus was delayed (44.2 vs 27.2 d; P < 0.01) in cows of DES-2100 micrograms group. All GnRH treatments elevated plasma P4 concentrations, and the highest P4 responses were observed in the DES-700 micrograms and DES-2100 micrograms groups. The second follicular wave emerged earlier in GnRH-treated than in control cows (9.9 vs 12.8 d; P < 0.01). However, emergence of the third dominant follicle was delayed in cows of DES-2100 micrograms treatment (37.0 d) compared with DES-700 micrograms (22.2 d), Buserelin (17.8 d) or control (19.0 d). In conclusion, Deslorelin implants of 700 micrograms increased plasma P4 and LH concentrations and slightly delayed the emergence of the third dominant follicle. On the contrary, Deslorelin implants of 2100 micrograms drastically altered the P4 profiles and follicle dynamics.     

Subdermal contraceptive implants

Subdermal contraceptive implants involve the delivery of a steroid progestin from polymer capsules or rods placed under the skin. The hormone diffuses out slowly at a stable rate, providing contraceptive effectiveness for 1–5 years. The period of protection depends upon the specific progestin and the type of polymer. Advantages of progestin implants include long term contraceptive action without requiring the user's or provider's attention, low dose of highly effective contraception without the use of estrogen, and fertility is readily reversible after the removal of implants. The levonorgestrel implant Norplant R system is the only one that has been approved for distribution. The contraceptive efficacy of Norplant is the highest observed amongst the most effective methods with an annual pregnancy rate of 0.2 during the first and second year and 1.1 on the fifth year. Menstrual problems are the main reason for the discontinuation of Norplant and 9% of women stopped using it during the first year of treatment. Other implants are still under development trying to simplify the method by reducing the number of units and to introduce other progestins that may minimize side effects. Norplant-2 was designed to release the same dose of progestin from only two covered rods. Evaluation of 1400 women enrolled, indicates that over 2 years the cumulative pregnancy rate is below 0.5 per 100 women. There are three single implants under development: Nestorone, 3-Keto-desogestrel and Uniplant that are expected to be effective for 1–2 years. Phase II clinical trials with Nestorone have been completed and no pregnancies have been observed in 1570 woman-months of use. Bleeding irregularities occurred in 20–30% of the women but there were only four terminations because of bleeding problems. A multricentric study is ongoing with a newly designed 3-keto-desogestrel implant named Implanon, which releases approx. 60 μg/day of the hormone. The objectives of this study are to assess contraceptive efficacy, safety and acceptability of Implanon. Another multricentric study is ongoing with Uniplant, which releases nomegestrol acetate with a duration of action for only 1 year. The objectives of the trial are to study the endocrine profile of Uniplant users and to evaluate the efficacy and acceptability of the method.     

Sensitivity differences in reproductive/endocrine organs to chronically administered LHRH agonists in female rats

The acute and chronic effects of two LHRH agonists on reproductive endocrine target organs were examined in female rats. Animals were injected twice daily with [(ImBzl)-D-His6,Pro9-NEt]LHRH (histrelin) or [D-Trp6,Pro9-NEt]LHRH for 1, 3, 5, 7, 11 or 28 days at 1, 10, 100 or 1000 micrograms/kg/day beginning in the luteal phase. The responses observed with the two agonists were similar. An initial stimulatory phase was observed on the first day of treatment with substantial increases in serum LH and progesterone levels. A significant diminution of hormone response was seen by day 3. Only 1000 micrograms/kg abolished the pituitary LH response at later treatment periods. Estrous cyclicity, ovarian and uterine weight, and progesterone and estradiol levels were inhibited in a time and dose dependent manner. The results demonstrate target organ sensitivity differences. In contrast to the relatively high doses needed to inhibit the pituitary response and decrease ovarian weight, doses as low as 1 microgram/kg were sufficient to decrease uterine weight. If these findings extrapolate to humans, it may be that conditions in which the desired therapeutic action is suppression of uterine tissue, may be treated with lower doses of LHRH agonists than conditions requiring complete gonadal suppression.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

A comparison of the LH-releasing activities of LH-RH and its agonistic analogue buserelin in the ovariectomized rat

LH-RH and the potent agonistic analogue (D-Ser(But)6-des-Gly10)-LH-RH(1-9)-ethylamide (HOE-766 or buserelin) were at several doses either infused or injected intravenously in 5-weeks-ovariectomized rats, which had been treated with either 3 micrograms estradiol-benzoate (EB) or with oil, 24 h previously. Blood samples for assay of LH were taken during the subsequent 24 h. Pituitary glands were removed at the end of the experiments. Buserelin, when infused, was slightly more effective than LH-RH on releasing LH. When injected, however, buserelin was at the higher dose ranges increasingly more effective as an LH-releasing agent than LH-RH. EB-treatment increased the LH response of the pituitary gland to both peptides in an identical way. It was concluded that buserelin derives its high potency not from its intrinsic LH-releasing activity, which is only slightly greater than that of LH-RH, but from a longer duration of action.     

Influence of intravenously administered leptin on nitric oxide production, renal hemodynamics and renal function in the rat

We investigated the effect of leptin on systemic nitric oxide (NO) production, arterial pressure, renal hemodynamics and renal excretory function in the rat. Leptin (1 mg/kg) was injected intravenously and mean arterial pressure (MAP), heart rate (HR), renal blood flow (RBF) and renal cortical blood flow (RCBF), were measured for 210 min after injection. Urine was collected for seven consecutive 30-min periods and blood samples were withdrawn at 15, 45, 75, 105, 135, 165 and 195 min after leptin administration. Leptin had no effect on MAP, HR, RBF, RCBF and creatinine clearance, but increased urine output by 37.8% (0–30 min), 32.4% (31–60 min) and 27.0% (61–90 min), as well as urinary sodium excretion by 175.8% (0–30 min), 136.4% (31–60 min) and 124.2% (61–90 min). In contrast, leptin had no effect on potassium and phosphate excretion. Plasma concentration of NO metabolites, nitrites+nitrates (NO_x), increased following leptin injection at 15, 45, 75 and 105 min by 27.7%, 178.1%, 156.4% and 58.7%, respectively. Leptin increased urinary NO_x excretion by 241.6% (0–30 min), 552.6% (31–60 min), 430.7% (61–90 min) and 88.9% (91–120 min). This was accompanied by increase in plasma and urinary cyclic GMP. These data indicate that leptin stimulates systemic NO production but has no effect on arterial pressure and renal hemodynamics.     

Use of buserelin to induce ovulation in the cyclic mare

Inducing ovulation in a cyclic mare is often necessary. For this purpose, hCG has been used commonly, but the response can be reduced after successive administrations. The aims of this study were to test the effectiveness of buserelin in hastening ovulation in estrus mares, and its influence on fertility; and to investigate the effect of treatment on LH secretion. Five crossover trials were designed to compare the effect of two treatments: buserelin (40 microg in 4 doses i.v. at 12 h intervals) vs placebo (Experiments 1 and 2); buserelin 40 microg (in 4 doses i.v.) vs 20 microg (Experiment 3); buserelin (4 doses of 20 microg i.v.) vs hCG (1 dose of 2,500 IU i.v.) (Experiment 4); or buserelin (3 doses of 13.3 microg at 6 h interval) vs hCG (Experiment 5). In Experiment 2, blood samples were taken hourly until ovulation, for LH measurements. In Experiment 1, buserelin treatment significantly hastened ovulation. Reduction of the dose by half (Experiment 3) did not alter the effectiveness. In Experiments 4 and 5, buserelin was as effective as hCG in inducing ovulation between 24 and 48 h after initiation of treatment. Buserelin treatment induced a rise in LH concentration during the 48 h period of the experiment, and LH concentrations before ovulation were significantly higher in buserelin treated cycles than in placebo cycles. These experiments demonstrated the usefulness of two new protocols of administration of buserelin, as an alternative to hCG for induction of ovulation. One hypothesis explaining the mechanism of action is that the persistant rise in LH concentration could modify the ratio of biological/immunological LH, as it occurs physiologically, thereby hastening ovulation.     

Use of LHRH agonists in the treatment of anovulation in women with polycystic ovary syndrome

The long-acting agonist analogue of LHRH, Buserelin (Hoechst) has been used to suppress endogenous gonadotrophins prior to induction of ovulation with low dose human menopausal gonadotrophin (HMG) in women with clomiphene-resistant polycystic ovary syndrome (PCOS). The results have been compared with those in a similar group of patients treated with HMG alone. Buserelin (900-1,500 micrograms/day) was given intranasally to 11 women who thereafter received a total of 33 cycles of treatment with low-dose HMG. The control group comprised 16 women who received 40 cycles of HMG without Buserelin pretreatment. The ovulation rate was similar in the two groups: Buserelin + HMG 70%, HMG alone 68% and both groups showed a high rate of single follicle ovulation (52 and 63%, respectively). The threshold dose of gonadotrophin required to induce a single follicle was similar in the two groups. Premature elevation of LH in the late follicular phase was common in women who received HMG alone, but did not occur in any cycle in the patients receiving Buserelin pretreatment. In summary, these data show that pretreatment with an LHRH analogue prevents a premature LH surge but it remains to be determined whether this will have a significant bearing on the rate of successful pregnancy in women with PCOS.     

Enhanced luteinizing hormone release by luteinizing hormone-releasing hormone in incubating female turkeys (Meleagris gallopavo)

To determine what role pituitary responsiveness plays in the suppression of gonadotropin level during incubation in the turkey, the ability of the pituitary to release luteinizing hormone (LH) in response to luteinizing hormone-releasing hormone (LHRH) was compared in incubating, laying, and photorefractory birds. In all three groups, the i.m. injection of LHRH (4 micrograms/kg) increased serum LH levels; however, the LH response was markedly enhanced in the incubating turkeys as compared with the laying (6.6-fold increase over preinjection levels vs. 1.9-fold; p less than 0.05) or the photorefractory birds (9.7-fold vs. 3.1-fold; p less than 0.05). The LHRH-induced LH release was also determined in turkeys as they shifted from the laying to the incubating phase of the reproductive cycle. This response increased (p less than 0.05) in magnitude as the birds started to incubate. The high prolactin level of incubating turkeys does not have a depressing effect on LHRH-stimulated LH release; thus, impaired LH response to LHRH is not a mechanism involved in the diminished gonadotropin secretion of incubating turkeys.     

Distribution of luteinizing hormone-releasing hormone receptors in the rat ovary

The distribution of luteinizing hormone-releasing hormone (LHRH) receptors was studied in the adult rat ovary using autoradiography after injection of the stable LHRH agonist ¹²⁵I-labelled [D-Ser(TBU)⁶,des-Gly-NH₂¹⁰]LHRH ethylamide (Buserelin) and by radioreceptor assay using the same tracer. In intact cycling female rats, no differences in ovarian LHRH receptor levels could be observed between day diestrus I and day proestrus. Moreover, similar levels are observed in total ovarian homogenate, corpora lutea and the remaining ovarian tissue in adult animals treated with PMSG (pregnant mare's serum gonadotropins) and hCG (human chorionic gonadotropin). Radioautographic data show a comparable distribution of grains over theca interna and externa, granulosa and luteal cells. The present findings indicate the presence of LHRH receptors in both the interstitial and follicular cells throughout all stages of cellular differentiation.     

Influences of castration and testosterone on spring to summer changes in release of luteinizing-hormone-releasing hormone in rabbits.

Push-pull cannulae were implanted toward the tuberal region of the hypothalamus in ten intact New Zealand male rabbits. In the first experiment, rabbits were perfused at different times after castration: 5-10 days (n = 10), 22-31 days (n = 9) and 50-64 days (n = 8). The release, mean amplitude and mean frequency of luteinizing-hormone-releasing hormone (LHRH) signals from 37 perfusions in ten animals were analysed in intact rabbits and at different times after castration. No significant changes in release of LHRH and in amplitude were observed, but the frequency was significantly higher 22-31 days after castration than in intact rabbits (intact: 0.86 +/- 0.12; castrated: 1.20 +/- 0.13 pulses h-1, P < 0.035; n = 9). In Expt 2, testosterone and placebo Silastic capsules were implanted in the castrated rabbits. Perfusions were performed in the following four periods, defined by season and time after testosterone and placebo implants: (i) spring; before implants, (ii) late spring; 0-2 weeks after implants, (iii) summer solstice; 2-4 weeks after implants and (iv) summer; 4-6 weeks after implants. Castrated rabbits were perfused during spring; castrated rabbits with testosterone capsule implants were perfused during late spring, around summer solstice and in summer and castrated rabbits with placebo implants were perfused during periods (iii) and (iv). Castrated animals with placebo implants showed no significant changes in mean LHRH release and amplitude, although the frequency was significantly higher around the summer solstice period than in castrated rabbits perfused in the spring. In castrated rabbits with testosterone implants LHRH release was significantly higher in late spring than around the summer solstice and in the summer. In addition, the concentrations of LHRH in late spring were significantly higher than those of intact and castrated animals. In contrast, mean LHRH amplitude and frequency did not change. Mean amount of LHRH released and amplitude in castrated rabbits with testosterone implants were significantly lower around the summer solstice than in late spring or summer and compared with intact animals around summer solstice and in castrated rabbits in early spring. These data demonstrate that there were no significant changes in the mean amplitude and release of LHRH after castration from 5 and up to 64 days in rabbits with hypothalamic push-pull cannulae, in contrast to the well established dramatic effect of castration on gonadotrophin concentrations. However, there was a small, but significant, increase in the mean frequency of LHRH pulses 22-31 days after castration compared with values from intact rabbits.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pituitary response to LHRH, LH pulsatility and plasma melatonin and prolactin changes in ewe lambs treated with melatonin implants to delay puberty

Prepubertal ewe lambs were treated with empty or filled melatonin implants. The implants were placed s.c. at birth and pituitary responsiveness to various doses of LHRH, LH/FSH pulsatility and prolactin and melatonin secretion were examined at 10, 19, 28, 36 and 45 weeks of age. Control animals (N = 10) showed no consistent alteration in pituitary responsiveness to LHRH during development. Ewes treated with melatonin (N = 10) had puberty onset delayed by 4 weeks (P less than 0.03) but no effect of melatonin on LH or FSH response to LHRH injection was observed at any stage of development. In the control and melatonin-treated ewe lambs the responses to LHRH injection were lower during darkness than during the day at all stages of development. No consistent differences in LH or FSH pulsatility were observed between treatment groups or during development. Prolactin concentrations, however, failed to decrease at the time of puberty (autumn) in the melatonin-treated group. Melatonin-treated ewe lambs maintained normal rhythmic melatonin production which was superimposed on a higher basal concentration and showed the same increase in melatonin output with age as the control ewes. These results indicate that the delayed puberty caused by melatonin implants is not due to decreased pituitary responsiveness to LHRH or to dramatic changes in basal LH or FSH secretion.