Potent LHRH Agonist Research Articles

Mechanisms for altered reproductive function in female rats following neonatal administration of raloxifene.

Raloxifene is a non-steroidal selective estrogen receptor modulator (SERM) that mimics estrogenic activity on bone density and blood lipid concentration without uterotropic actions. Previous data from our laboratory indicated that, as is the case for estrogen, neonatal administration of raloxifene disturbed normal differentiation of the hypothalamic circuitries governing the gonadotropic axis. In contrast, raloxifene did not act in the same way as estrogen does on the neuronal systems controlling sexual receptivity in the female rat. At present, however, the mechanisms for these organizing effects of raloxifene are not completely elucidated. To analyze this phenomenon, female rats were injected daily with raloxifene (50, 100, 250 or 500 microg/rat per day) between days 1 and 5 of age. On day 23, hypothalamic gonadotropin-releasing hormone (LHRH) mRNA expression was assessed, and pituitary and plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured in basal and LHRH-stimulated conditions. In addition, LH and FSH responses to ovariectomy were evaluated in raloxifene-treated females. Finally, we monitored the ability of neonatal administration of a potent LHRH agonist ([d-Ala(6),d-Gly(10)]-LHRH ethylamide; 0.01 microg/kg per 12 h on days 1-5) to counteract the effects of raloxifene. Our analyses demonstrated that prepubertal rats (23-day-old females) treated neonatally with raloxifene showed decreased hypothalamic LHRH mRNA expression levels, reduced pituitary content of LH and FSH, reduced basal and LHRH-stimulated LH secretion in vivo and in vitro, and decreased response to ovariectomy. In addition, adult females treated neonatally with raloxifene showed anovulation and reduced serum LH levels; these effects were not prevented by the simultaneous administration of a LHRH agonist. In conclusion, our data demonstrate that neonatal administration of raloxifene can disrupt the programming of hypothalamic-pituitary-ovarian axis function. Reduced LH secretion, under basal and LHRH-stimulated conditions and after ovariectomy, is probably related to decreased LHRH expression, reduced pituitary LH content and/or decreased pituitary responsiveness to hypothalamic LHRH.

Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression.

Luteinizing hormone-releasing hormone (LHRH) is the key hormone in the control of reproductive functions. In recent years, it has become evident that LHRH might act as a growth modulatory factor in tumors of the reproductive system. We have shown that in prostate cancer cells LHRH is expressed, together with its receptors, to negatively regulate cell proliferation. In these cells, LHRH acts as an antimitogenic factor through the activation of the Gi-cAMP intracellular signaling pathway. More recently, we investigated whether an LHRH-based autocrine system might also be expressed in tumors that are not classically related to the reproductive tract, such as melanoma. Malignant melanoma is known to be characterized not only by a high proliferation rate, but also by an aggressive metastatic behavior. We have demonstrated that both LHRH and LHRH receptors are expressed in human melanoma cells (BLM and Me15392). Activation of LHRH receptors by means of a potent LHRH agonist (Zoladex) significantly inhibited cell proliferation. The LHRH agonist also reduced the ability of melanoma cells to invade a reconstituted basement membrane (Matrigel) and to migrate in response to a chemotactic stimulus. These data indicate that: (a) in prostate cancer cells the LHRH receptor is coupled to a Gi-cAMP signal transduction pathway; (b) LHRH and LHRH receptors are also expressed in tumors that are not classically related to the reproductive system, such as melanoma; in melanoma cells, LHRH might act as an inhibitory factor on both cell proliferation and metastatic behavior. It is suggested that, in melanoma, LHRH receptors might represent a diagnostic marker and a possible molecular target for new therapeutic approaches for this pathology.

Characterization of the solution conformations of leuprolide acetate.

Leuprolide acetate (pGlu-His-Trp-Ser-Tyr-d-Leu-Leu-Arg-Pro-NHEt), a potent LHRH agonist in wide clinical use, was characterized conformationally by NMR and circular dichroism. It displayed quite different preferred conformations under different solution conditions: two low population beta-turns in water, a nascent helix in TFE/water at low pH, and a high population beta-turn in TFE/water at slightly acidic pH. The pH-related conformational change in TFE/water is attributed to the pK(a) of the acetate counterion, not to ionizable groups on the peptide. None of these conformations are in exact agreement with previous computational predictions.

Locally Expressed LHRH Receptors Mediate the Oncostatic and Antimetastatic Activity of LHRH Agonists on Melanoma Cells

Malignant melanoma is a tumor known for its uncontrollable growth and aggressive metastatic behavior. The mean survival time for patients with a metastatic melanoma is estimated to be less than 6 months, tumor cells being refractory to the conventional chemotherapy. A better understanding of the mechanisms regulating melanoma growth and progression might help increase the number of therapeutic options for this pathology. In this paper, we have shown that LHRH receptors are present in the BLM melanoma cell line, both at mRNA and at protein level; a potent LHRH agonist (LHRH-A; Zoladex) binds to these receptors with high affinity. BLM cells also express the mRNA for LHRH, indicating the presence of an autocrine LHRH-based system in melanoma cells. The treatment of BLM cells with LHRH-A dose-dependently inhibited cell proliferation; this effect was found to be specific because it was completely abrogated by the simultaneous treatment of the cells with a LHRH antagonist. Similar observations could be obtained in another melanoma cell line (Me15392). The activation of LHRH receptors, by means of LHRH-A, also reduced the ability of melanoma cells to invade a reconstituted basement membrane (Matrigel) and to migrate through a Boyden’s chamber in response to a chemotactic stimulus. These data represent the first report that 1) LHRH and LHRH receptors are expressed in melanoma tumor cells; and 2) the activation of tumor LHRH receptors reduces both the proliferation and the metastatic potential of melanoma cells. It is suggested that the expression of LHRH receptors might represent a new diagnostic marker for the detection and progression of melanoma. These receptors might also be considered as a possible molecular target for a hormone-based therapeutic approach to this tumor.

Locally expressed LHRH receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells.

Malignant melanoma is a tumor known for its uncontrollable growth and aggressive metastatic behavior. The mean survival time for patients with a metastatic melanoma is estimated to be less than 6 months, tumor cells being refractory to the conventional chemotherapy. A better understanding of the mechanisms regulating melanoma growth and progression might help increase the number of therapeutic options for this pathology. In this paper, we have shown that LHRH receptors are present in the BLM melanoma cell line, both at mRNA and at protein level; a potent LHRH agonist (LHRH-A; Zoladex) binds to these receptors with high affinity. BLM cells also express the mRNA for LHRH, indicating the presence of an autocrine LHRH-based system in melanoma cells. The treatment of BLM cells with LHRH-A dose-dependently inhibited cell proliferation; this effect was found to be specific because it was completely abrogated by the simultaneous treatment of the cells with a LHRH antagonist. Similar observations could be obtained in another melanoma cell line (Me15392). The activation of LHRH receptors, by means of LHRH-A, also reduced the ability of melanoma cells to invade a reconstituted basement membrane (Matrigel) and to migrate through a Boyden's chamber in response to a chemotactic stimulus. These data represent the first report that 1) LHRH and LHRH receptors are expressed in melanoma tumor cells; and 2) the activation of tumor LHRH receptors reduces both the proliferation and the metastatic potential of melanoma cells. It is suggested that the expression of LHRH receptors might represent a new diagnostic marker for the detection and progression of melanoma. These receptors might also be considered as a possible molecular target for a hormone-based therapeutic approach to this tumor.

Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D-Trp(6)]LHRH on the basal and EGF-induced cell proliferation and the metastasis-associated properties in A431 human epidermoid carcinoma. [D-Trp(6)]LHRH time-dependently inhibited the basal and EGF-stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D-Trp(6)]LHRH decreased the basal and EGF-induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D-Trp(6)]LHRH did not cause detectable changes in basal and EGF-stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D-Trp(6)]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1beta-converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D-Trp(6)]LHRH treatment for 48 hr, which suggests that limited exposure to [D-Trp(6)]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D-Trp(6)]LHRH on metastasis-associated properties in A431 cells. [D-Trp(6)]LHRH reduced both basal and EGF-stimulated secretion of MMP-9 and MMP-2. In addition, [D-Trp(6)]LHRH suppressed the basal and EGF-induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D-Trp(6)]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis-associated properties of A431 cancer cells. Our work suggests that [D-Trp(6)]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms.

Acute N-Methyl-D,L-Aspartate Administration Stimulates the Luteinizing Hormone Releasing Hormone Pulse Generator in the Ovine Fetus

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130–142 days of gestation; term 147 ± 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 ± 0.36 ng/ml) and oFSH (1.21 ± 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 μg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp⁶Pro⁹NEt-LH-RH (10 μg/day i.v. × 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D,L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10^–10 to 10^–6 M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10^–8 M; p < 0.0004) and oFSH (10^–8 M; p < 0.0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

Protective effect of leuprolide acetate on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinogenesis in rats

The anti-carcinogenic effect of TAP-144-SR biodegradable microcapsules of copoly (DL-lactic/glycolic acid) copolymer containing a potent LHRH agonist, TAP-144 (D-Leu6-(des-Gly10-NH2)-LHRH ethylamide, leuprolide acetate) was investigated in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumours. At 50 days of age, all rats were given 20 mg doses of DMBA by gastric intubation. Rats on the Initiation Group were given a single s.c. injection of TAP-144-SR (3 mg/kg) 1 week before DMBA treatment, and rats in the Promotion Group were given repeated s.c. injections every fourth week from 1 week after DMBA treatment until the end of the experiment. In the Initiation Group, tumour incidence and multiplicity were reduced to 20.8% and 15.0% of the Control Group values, respectively. In the Promotion Group, tumour incidence and multiplicity were also reduced to 17.7% and 7.9%, respectively. It was suggested that TAP-144-SR may be a potential candidate as an agent for hormonal chemoprevention in breast cancer.

Drug delivery using biodegradable microspheres

Rational delivery systems for leuprorelin acetate, a potent LHRH agonist, have been achieved by developing a microsphere system using biodegradable polymers, poly(lactic/glycolic acid) (PLGA) and polylactic acid, which sustainedly release the drug depending on the biodegradation of polymer used and persistently suppress steroidogenesis for over one and 3 months, respectively, following a single injection. To produce these systems we established a novel microencapsulation technique, the in-water drying method, and microspheres with a high trap ratio and small initial burst were obtained. A microsphere system of TRH prepared using PLGA could also continuously release the drug for 2 or 4 weeks. Using these systems effectively reduced the required dose compared with that needed with daily injection due to more continuous receptor hits on the target organs and could improve patient compliance. Chemoembolization using PLGA microspheres containing an angiogenesis inhibitor, TNP-470, resulted in dramatic regression of VX-2 carcinoma in rabbits. The microsphere system using biod-egradable polymers is very useful in designing controlled release delivery and targeted delivery to attain potent and rational therapy.

Experience with a potent LH-RH agonist, Buserelin, alone and in combination with testosterone for antispermatogenic activity, reversibility and toxicity in langur monkey

Chronic intermittent treatment of LH-RH superagonist Buserelin alone or in combination with testosterone enanthate were given to adult male langurs for 90 days to evaluate antispermatogenic activity of alone and combination therapy, maintenance of normal androgenicity, possible toxic effects of agonist treatment, related side effects of testosterone supplementation and complete reversibility of the procedure. A gradual decrease in sperm count was recorded in both treatment groups, along with reduced motility and vitality of the spermatozoa. In the combination group, oligospermia was achieved in 4 out of 5 animals, whereas, only 2 animals became oligospermic in the agonist alone group. Significant decrease in serum testosterone levels along with impaired libido and other testosterone withdrawal symptoms were observed in the Buserelin alone group, conversely normal testosterone levels and libido were observed in the combination group. An elevation in haematological variables and serum total protein concomitant with a slight gain in body weight of the animals were recorded in the combination group; these changes were not encountered in the agonist alone group. Reversibility of all the altered parameters to control range was observed in both treatment groups following 75 to 90 days of treatment withdrawal.

Intratesticular Control Of Steroidogenesis

Men are men because of testosterone, and the effects of this hormone are evident in virtually every organ or tissue in a normal adult man. Because testosterone is a classical endocrine hormone with effects throughout the body, many endocrinologists will have failed to realize that, in terms of its effects on fertility, the primary role of testosterone is not endocrine but paracrine, as it is the local action of testosterone on spermatogenesis which determines fertility. When looked at from this perspective it is logical to question the traditional endocrine teaching which states that testosterone secretion is controlled solely by pituitary luteinizing hormone (LH). Certainly, the levels of testosterone and LH in peripheral blood show a close relationship, but it is not the level of testosterone in peripheral blood which determines fertility. If the primary role of testosterone is within the testis, then it is not unreasonable to expect that local mechanisms may exist to regulate the intratesticular levels of this steroid. The object of this review is to examinecritically the evidence for such mechanisms, their possible nature and their physiological and clinical significance (if any). Before reviewing the data on intratesticular control of steroidogenesis it is essential to place it in perspective. To do this, three principal questions need to be answered: (I) Why might intratesticular mechanisms for local control of testosterone production be required? (2) What is the role (or roles) of testosterone within the testis? A proper understanding of how testosterone controls spermatogenesis is essential if the local regulation of testosterone production is to be evaluated from a physiological perspective. (3) What are the constraints or problems relating to studies on the intratesticular control of steroidogenesis? It is essential to understand the potential limitations of studies on this subject so that a reasoned interpretation of the results can be made. Most relevant data is derived from studies in uitro, and the relationship of such data to the situation in uivo is often difficult to establish.

Pharmacological effects of the continuous administration of an LHRH agonist in the ageing male rat: comparison with orchidectomy

The age-related changes in tissue response to chronic treatment for 1 month with a potent LHRH agonist were investigated in the ageing male rat, and the observed pharmacological effects were compared with orchidectomy. In both young (4 months) and old (22 months) rats, treatment resulted in a significant decrease in the weights of prostates and testes, a decrease in plasma LH and testosterone levels, a loss of LH receptors in the testes and in a complete depletion of prostatic nuclear androgen receptors, reaching levels observed after castration. In young rats, treatment with an LHRH agonist or orchidectomy induced a three- or sixfold increase in prostatic creatine kinase (CK) activity which may have been induced by the local stimulatory effect of oestradiol arising from the conversion of precursor steroids secreted by the adrenal. On the other hand, in old rats, 7 days after orchidectomy or after treatment with an LHRH agonist a twofold increase or no change was induced in prostatic CK activity respectively. SDS gel electrophoresis patterns of cytosolic prostatic proteins of young rats treated with an LHRH agonist or young rats orchidectomized 7 days previously revealed the presence of several intensified proteins, two of them having apparent molecular weight of 67 kDa and 43 kDa, whereas in the old rats treated with LHRH agonist or old rats castrated 7 days previously, these two proteins were not intensified. The results of this study confirmed that continuous treatment with an LHRH agonist to young and old rats induces medical castration since the pharmacological effects observed were the same as those induced with surgical castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Histological Studies on the Therapeutic Effect of Sustained-Release Microspheres of a Potent LHRH Agonist (Leuprorelin Acetate) in an Experimental Endometriosis Model in Rats.

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.

Luteinizing Hormone-Releasing Hormone (LHRH) Agonist Restoration of Age-Associated Decline of Thymus Weight, Thymic LHRH Receptors, and Thymocyte Proliferative Capacity

The presence of specific LHRH-binding sites within the rat thymus gland and the ability of LHRH and its agonistic and antagonistic analogs to directly modulate thymus function prompted us to study the possible changes in the number of thymic LHRH-binding sites during aging-induced physiological immunosenescence. Moreover, the effects of chronic treatment of aging rats with a potent LHRH agonist (LHRH-A) on thymic LHRH receptors, thymus weight and histology, as well as thymocyte proliferative capacity were assessed. For comparison, the effects of castration on the same parameters was also investigated. The process of aging is accompanied by a sharp reduction in LHRH-A-binding sites within the thymus gland of both female and male rats. Starting at 7 months of age, a 50% decrease in thymic LHRH-A binding was followed, at 11-13 months of age, by a nearly 65% inhibition of receptor numbers. In 16- to 19-month-old rats, LHRH-A binding was almost completely lost. Thymus weight was 30% reduced in 7-month-old animals, while a 50% reduction in thymic size was reached at 11 months of age in males and 13 months in female rats. A further decrease in thymic mass was observed at 16 and 19 months. Chronic (45-day) treatment of aging (15-16 months old) female and male rates with the potent LHRH-A, [D-Trp6,Des-Gly10]LHRH-N-ethylamide, reversed the age-related decreases in both thymus weight and thymic LHRH-binding sites. Similarly, surgical removal of testicular hormones by castration restored thymus weight and increased LHRH-A binding in the thymus of aged rats. While thymus histology in 3-month-old rats was characterized by a clear demarcation of cortical and medullary regions, only thymic remnants were present in 16- to 17-month-old animals. Castration of old rats resulted in a partial restoration of thymic structure, while chronic treatment of aging rats with the LHRH-A produced a homogeneous organization of both cortical and medullary compartments accompanied by a marked increase in the width of the cortical layer, densely packed with lymphocytes. While the process of aging was accompanied by an almost complete loss of the proliferative response of thymocytes to optimal concentrations of the mitogen Concanavalin-A, thymocyte cultures from old rats treated with LHRH-A or from castrated animals, displayed significantly greater proliferative responses. Furthermore, the combination of both manipulations resulted in a further significant increase in thymocyte proliferative capacity.(ABSTRACT TRUNCATED AT 400 WORDS)

Luteinizing Hormone-Releasing Hormone-Binding Sites in the Rat Thymus: Characteristics and Biological Function

The present study was designed to explore the effects of LHRH and its agonists on immune system function. As a first step, to identify a putative site of action, the very potent and stable LHRH agonist (LHRH-A), [D-Ser(TBU6)] des-Gly10-LHRH ethylamide (buserelin), was used as an iodinated ligand to characterize LHRH receptors in a membrane preparation of rat thymus, a key organ of the immune system. The effects of LHRH and LHRH-A were then investigated on the proliferative capacity of rat thymocytes exposed in vitro to a mitogen and on ornithine decarboxylase specific activity. In addition, to determine whether LHRH-A treatment in vivo might directly influence thymic function, we treated hypophysectomized (hypox) rats with a moderately high dose of LHRH-A for a period of 2 weeks, and thymocyte mitogenic capacity, thymus weight, and the histological and functional appearance of the thymus were then assessed. Specific binding of LHRH-A to rat thymic membrane preparations is a saturable process, depending on both time and temperature of incubation, but differs markedly from binding to the rat pituitary or ovarian LHRH receptor in its low binding affinity. Binding is optimal in the absence of chelating agents (EDTA) or divalent metal ions, and increases linearly with increasing protein concentration. Binding is specific for LHRH, LHRH-A, and antagonists. Both the C-terminal amide and N-terminal regions of the LHRH molecule were required for binding, and amino acid substitutions at position 6 markedly enhanced and at position 8 markedly reduced binding potencies in rat thymic tissue. A number of peptides, proteins, and other agents had no effect on the specific binding of LHRH-A to thymic membrane preparations. The binding affinity (Ka) of the membrane receptor of the rat thymus for the LHRH superagonist buserelin was 8.4 x 10(8) M-1, while a higher binding affinity (Ka = 2.8 x 10(9) M-1) was calculated for the ovarian LHRH-binding site. Preincubation of rat thymocytes with LHRH-A for 20 h induced a significant dose-dependent increase in the proliferative response to the mitogen Concanavalin-A, monitored by [3H]thymidine incorporation. Using native LHRH, it was also possible to elicit stimulatory effects on the same parameter, although much higher concentrations were required than with LHRH-A. Furthermore, simultaneous addition of a LHRH antagonist, abolished the LHRH effect on thymocytes. Ornithine decarboxylase specific activity under lectin stimulation was also significantly increased by LHRH-A in cultures of rat thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens modulate epidermal growth factor receptor levels in the rat ventral prostate

In order to further understand the factors which influence the normal or pathologic growth of the prostate, we have characterized the receptor for epidermal growth factor (EGF) in the rat ventral prostate and have studied the hormonal regulation in this receptor. EGF binds to a single class of saturable, high affinity binding sites in total prostatic homogenate. Scatchard analysis of the binding data reveals an apparent dissociation constant ( K d ) of 0.93 ± 0.08 nM and a number of sites of 4.01 ± 0.24 fmol per mg protein. Among the peptides tested, only native EGF can displace bound [ 125I]EGF. Castration stimulates the concentration of prostatic EGF receptors from 25.5 ± 3.0 to 43.4 ± 5.4 fmol/100 mg tissue in intact and castrated animals, respectively ( P < 0.01). Treatment of castrated rats with dihydrotestosterone (DHT) inhibits the rise in prostatic EGF receptor concentration induced by orchiectomy, while estradiol, progesterone or the dopaminergic agonist CB-154, have no effect. Combined administration of DHT with the other above-mentioned steroids or CB-154 does not modify the inhibition of prostatic EGF receptor concentration induced by the androgen in castrated animals. When the data are expressed as changes in EGF receptor number in the total prostate, DHT treatment reverses the inhibitory effect induced by castration and yields an EGF binding capacity comparable to that measured in intact animals. Chronic treatment with a pure antiandrogen or a potent LHRH agonist (LHRH-A) alone has no significant effect on EGF receptor concentration in prostatic tissue, although, secondary to a reduction in prostatic weight, total prostatic EGF binding capacity is reduced following antiandrogen or LHRH-A treatment. When the agonist, however, is administered in combination with the antiandrogen, the concentration of prostatic EGF receptors rises from 12.1 ± 1.3 fmol/100 mg tissue in intact animals to 35.8 ± 4.0 fmol/100 mg tissue in animals receiving the combined treatment with the highest (9 mg/day) dose of antiandrogen. This degree of stimulation (about 300%) is comparable to the stimulation induced by castration. While both prostatic EGF binding capacity and tissue weight are strongly modulated by androgens, the effect on EGF receptors is of smaller magnitude, thus indicating a relative stability of this parameter in the prostatic tissue. The presence of specific and high affinity receptors for EGF in the rat ventral prostate and the

The antiandrogen anandron potentiates the castrating effect of the LH-RH agonist buserelin in the rat.

When buserelin (0.04-25 micrograms/kg/day), a potent LH-RH agonist, was administered s.c. daily for 15 days to male rats, prostate weight decreased after a transient increase in the first days of treatment but never as much as after orchidectomy, since testosterone secretion was never totally suppressed. The combination of the pure nonsteroid antiandrogen Anandron (20 mg/kg/day) with even low doses of buserelin led to an immediate decrease in prostate weight that was complete at 15 days. This could be explained (a) by an additivity of effects: inhibition by Anandron of the action of residual testosterone unsuppressed by buserelin on the prostate and prevention by buserelin of the rebound testosterone increase induced by Anandron: (b) by a potentiation by Anadron of the direct castrating effect of buserelin on the testis since testis weight and testosterone secretion were lowered to a greater extent by the combination than by buserelin alone. Anandron might increase the sensitivity of LH-RH receptors to LH-RH agonist in the testes, as has been shown in the pituitary. In contrast, the combination of the antiandrogenic steroid progestin, cyproterone acetate, with buserelin never potentiated the castrating effect of buserelin on testis weight and testosterone and only partially potentiated prostate atrophy. If such actions also exist in the human, the addition of Anandron to LH-RH agonist treatment would not only counter flare-up and adrenal androgen effects on the prostate but would also lower the doses of peptide or the time required for chemical castration.

Evidence for a negative ultrashort loop feedback mechanism operating on the luteinizing hormone-releasing hormone neuronal system.

The present studies were designed to determine whether an ultrashort loop feedback mechanism is involved in the regulation of LHRH secretion. Daily administration of a highly potent LHRH agonist (LHRH-AGO; [D-Ala6,Des-Gly10] LHRH ethylamide) immediately after orchidectomy (ORDX) significantly attenuated the rise of plasma LH from days 2 through 10 after ORDX. Concomitantly with the diminished LH rise after ORDX, a significant increase in LHRH content in the arcuate nucleus was observed in LHRH-AGO-treated rats. Measurement of LHRH levels in hypophyseal portal blood in rats 10 days after ORDX combined with daily agonist treatment revealed a significant decrease in LHRH values in portal plasma compared with those in orchidectomized controls. Arcuate nuclei-median eminence (ME) fragments obtained from ORDX rats treated in vivo with LHRH-AGO for 5 days showed a decreased basal secretion of LHRH and a diminished response to K+ stimulation compared with the release from fragments obtained from ORDX saline-treated controls. To evaluate whether a tonic LHRH inhibitory activity operates within the ME, additional experiments were performed in which ME fragments were incubated in vitro in the presence of a potent LHRH antagonist [( D-pGlu1,D-Phe2,D-Trp3,6]LHRH). The antagonist significantly enhanced the basal secretion of LHRH in a dose-dependent manner. The latter results suggest that LHRH antagonists may enhance LHRH release, perhaps by interacting with LHRH receptors playing an inhibitory role on the endogenous secretion of the decapeptide. These observations strongly suggest a tonic inhibitory or modulatory role of LHRH neurons in the regulation of their own function.

Effects of a potent LHRH-agonist on the pituitary gonadal axis with and without testosterone substitution.

In this study we investigated the effect of low and high dosages of a potent LHRH agonist on the pituitary-gonadal axis with special consideration to the effect on the tubular compartment of the testis. Included were 3 treatment groups: the probands in Group I were treated with 3 X 50 micrograms HOE 766/week intranasally for 5 months; in Group II with 3 X 100 micrograms HOE 766 intranasally/day for 6 months and in Group III with 3 X 200 micrograms HOE 766 intranasally plus 5 mg fluoxymesterone orally/day for 5 months. With the low dose (Group I) no changes in the seminal parameters measured could be observed whereas LH and FSH levels increased in plasma, testosterone showed no change compared to pretreatment values. When high dosages/day of a potent LHRH agonist were administered without androgen replacement (Group II) pronounced decrease of LH and FSH took place, the testosterone plasma levels approached the female range. Spermatogenesis was arrested. The agonist plus androgen replacement (Group III) counteracted the suppression of spermatogenesis.

Lack of correlation between fertility and sperm numbers in male rats treated with histrelin, a potent LHRH agonist.

Adult male rats were treated daily for up to 8 weeks with histrelin, [(ImBzl)-D-His6, Pro9-NEt]LHRH, to study the antifertility effects of this LHRH agonist. Although serum testosterone concentrations and testicular sperm numbers were significantly decreased by weeks 2 and 4 respectively, a reduction in fertility, as judged by the mean number of fetuses per mated female, was not observed until the sixth week of treatment. Furthermore, the number of spermatozoa in the cauda epididymidis of treated rats did not decrease below initial control levels at any time during the study and full fertility returned within 4 weeks after histrelin treatment was stopped. Thus, the lack of correlation between fertility and testicular and epididymal sperm numbers suggests that the antifertility effects of LHRH agonists are not due solely to reduced sperm numbers, but also result from androgen deficiency.