Suppression Of LH Research Articles

Potential of Norethisterone Enanthate for Male Contraception: Pharmacokinetics and Suppression of Pituitary and Gonadal Function

OBJECTIVE Gestagens are known to suppress gonadotrophins in women and are currently also under investigation for the development of hormonal male contraceptives. The aim of the study was to assess the potential of norethisterone enanthate (NETE) for male contraception. DESIGN AND MEASUREMENTS The suppressive effect of a single injection of 200 mg NETE on serum gonadotrophins, serum testosterone, lipids, spermatogenesis, well-being and sexual function was evaluated in seven healthy men. RESULTS In this single dose study treatment was well tolerated by all volunteers. NETE led to a rapid, profound and significant suppression of serum LH (day 6 - day 10), FSH (day 2 - day 29), testosterone (day 1 - day 29 and day 35) and SHBG (day 6 - day 35). At study end sperm counts were significantly suppressed. Numbers of spontaneous erections (day 17, 23 and 26), number of sexual fantasies (day 20 and 23) as well as libido (day 20 and 26) were significantly decreased compared to baseline. All other parameters including lipids, augmented glucose, testicular volume and well-being showed no significant alterations. CONCLUSION Because of its strong, rapid and sustained suppression of serum FSH and testosterone norethisterone enanthate offers great potential for hormonal male contraception if combined with testosterone esters.

Pharmacodynamic effects and plasma pharmacokinetics of single doses of cetrorelix acetate in healthy premenopausal women

Objective: To investigate the pharmacodynamic effects and plasma pharmacokinetics of single subcutaneous doses of cetrorelix acetate in healthy premenopausal women. Setting: Phase I clinical research unit. Patient(s): Healthy, premenopausal women aged 19 to 35 years. Intervention(s): Single subcutaneous morning doses of cetrorelix acetate (1, 3, or 5 mg peptide base) were investigated in a randomized, single-blind, placebo-controlled, parallel-group design. After a control cycle, 36 women received cetrorelix acetate (12 per dose) and 12 received placebo on the eighth individual cycle day. Transvaginal ultrasound was performed, and blood samples for LH, FSH, E 2 were collected during both cycles and for pharmacokinetics up to 168 hours after dosing. The serum hormone levels were determined by electrochemicoluminescence immunoassay and plasma cetrorelix concentrations by radioimmuno assay. Results: Cetrorelix acetate administration led to a rapid, marked, and reversible suppression of serum LH, E 2, and to a lesser extent FSH concentrations. The median intra-individual shifts between treatment and control cycle were −1.0, 4.0, 8.0, and 9.5 days for serum LH maximum and −1.0, 4.5, 7.0, and 10.0 days for ovulation following placebo or 1, 3, and 5 mg cetrorelix acetate, peptide base, respectively. The area under the concentration-time curve (AUC) and peak cetrorelix concentrations in plasma (C max) increased proportionally with dose. Conclusions: Cetrorelix acetate showed pronounced and reversible LH and E 2 suppression and a dose-dependent postponement of LH surge and ovulation after single subcutaneous administrations to healthy premenopausal women. Dose proportionality over the range of 1 mg to 5 mg cetrorelix acetate, peptide base was demonstrated.

Is GnRH self-priming an obligatory feature of the reproductive cycle?

Insufficient suppression of LH (premature elevation) and FSH (prolonged release) give rise to blood concentrations which may cause damaging effects on oocyte viability and too many follicles respectively. During the surge, LH rises from low to high threshold values to initiate processes from initiation of the resumption of oocyte meiosis to the induction of ovulation. In general, it is thought that a dramatic increase in LH concentration is required to attain the high threshold for ovulation. A self-priming mechanism, by which gonadotrophin-releasing hormone (GnRH) enhances the LH (and FSH) responses to its own action, was thought to be responsible. However, normal LH surges in rats consist of <2-7% of the maximal pituitary releasing capacity. The physiological roles of LH and FSH favour a control mechanism that restrains their blood concentrations during most of the cycle. Ovarian proteins, e.g. inhibin and putative gonadotrophin-surge-inhibiting factor/attenuating factor (GnSIF/AF), are involved in this process. We argue that the increased pituitary LH responsiveness during the mid-cycle surge is not the result of a self-priming process that 'dramatically' increases the LH releasing capacity of the pituitary gland. This is probably due to elimination by GnRH of the inhibitory action of the putative ovarian proteins GnSIF/AF.

Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats.

A single intraperitoneal injection of lipopolysaccharide (LPS) causes a biphasic suppression of testicular steroidogenesis in adult rats, with inhibition at 6 h and 18-24 h after injection. The inhibition of steroidogenesis is independent of the reduction in circulating LH that also occurs after LPS treatment, indicating a direct effect of inflammation at the Leydig cell level. The relative contributions to this inhibition by intratesticular versus systemic responses to inflammation, including the adrenal glucocorticoids, was investigated in this study. Adult male Wistar rats (eight/group) received injections of LPS (0.1 mg/kg i.p.), dexamethasone (DEX; 50 microg/kg i.p.), LPS and DEX, or saline only (controls), and were killed 6 h, 18 h and 72 h later. Treatment with LPS stimulated body temperature and serum corticosterone levels measured 6 h later. Administration of DEX had no effect on body temperature, but suppressed serum corticosterone levels. At the dose used in this study, DEX alone had no effect on serum LH or testosterone at any time-point. Expression of mRNA for interleukin-1beta (IL-1beta), the principal inflammatory cytokine, was increased in both testis and liver of LPS-treated rats. Serum LH and testosterone levels were considerably reduced at 6 h and 18 h after LPS treatment, and had not completely recovered by 72 h. At 6 h after injection, DEX inhibited basal IL-1beta expression and the LPS-induced increase of IL-1beta mRNA levels in the liver, but had no effect on IL-1beta in the testis. The effects of DEX on IL-1beta levels in the liver were no longer evident by 18 h. In LPS-treated rats, DEX caused a significant reversal of the inhibition of serum LH and testosterone at 18 h, although not at 6 h or 72 h. Accordingly, DEX inhibited the systemic inflammatory response, but had no direct effect on either testicular steroidogenesis or intra-testicular inflammation, at the dose employed. These data suggest that the inhibition of Leydig cell steroidogenesis at 6 h after LPS injection, which was not prevented by co-administration of DEX, is most likely due to direct actions of LPS at the testicular level. In contrast, the later Leydig cell inhibition (at 18 h) may be attributable to extra-testicular effects of LPS, such as increased circulating inflammatory mediators or the release of endogenous glucocorticoids, that were inhibited by DEX treatment. These data indicate that the early and late phases of Leydig cell inhibition following LPS administration are due to separate mechanisms.

Effects of paint thinner exposure on serum LH, FSH and testosterone levels and hypothalamic catecholamine contents in the male rat.

We have investigated the effects of thinner inhalation on serum LH, FSH and testosterone levels together with changes in hypothalamic catecholaminergic system in the male rat. A control group inhaled normal air ventilation. The remaining animals were divided into two groups and exposed to paint thinner in a glassy cage for 15 or 30 d. Toluene concentration (the largest constituent in thinner, 66%) was set at 3000 ppm in the inhalation air. At the end, all animals were decapitated and blood samples obtained. Serum LH and FSH levels were measured by RIA and testosterone by enzyme immunoassay. Following removal of brains on dry ice, medial preoptic area, suprachiasmatic nucleus, median eminence and arcuate nucleus were isolated by micropunch technique. Noradrenaline, 3,4-dihydroxyphenylglycol (DHPG) and dopamine concentrations of these hypothalamic areas were determined by HPLC-ECD. Fifteen-day thinner inhalation significantly suppressed serum LH and testosterone levels in parallel (p<0.001) compared to control group values (LH: 0.77+/-0.07; testosterone: 2.67+/-0.39). Thirty-day exposure markedly decreased LH levels (p<0.001), but surprisingly had no significant effect on testosterone. Serum FSH levels were not significantly altered in either group. Thinner inhalation for 15 or 30 d did not cause any significant change in noradrenaline, DHPG or dopamine concentrations in the hypothalamic regions examined (except in the arcuate nucleus). These results suggest that paint thinner has an anti-gonadotropic effect and may cause long-term endocrine disturbances in the male. It is thought that the hypothalamic catecholaminergic system is not involved in thinner inhibition of LH and testosterone secretion.

Hormonal and ovarian responses to a 5-day progesterone treatment in anoestrous dairy cows in the third week post-partum

Primiparous cows with low body condition at calving have an extended anovulatory period. Induction of ovulation and oestrus is possible with progesterone treatment but the response to this treatment differs between Friesian and Jersey breeds. The objective of this study was to describe changes in pulsatile LH secretion and the synchrony of developing ovarian follicles that occur during a progesterone treatment period of 5 days in primiparous anovulatory cows. The experimental model compared the progesterone treatment with spontaneous post-partum changes as well as a breed comparison in a factorial design. Thirty-six cows (Jersey n=19 and Friesian n=17) were managed to calve with a low body condition score (BCS<4.5). Daily changes in ovarian follicle size were observed with transrectal ultrasonography in each cow from 8 days post-partum. Thirty of these cows were diagnosed to be anovulatory at 12–18 days post-partum (day 0) and allocated to a treatment ( n=16) or a control group ( n=14), balanced for breed. Each treated cow had a progesterone-releasing controlled internal drug-releasing (CIDR) device inserted vaginally for 5 days while control cows were left untreated. Changes in plasma LH concentrations were measured with intensive blood sampling over 8 h on days −1, 1, and 4. Blood samples were also collected daily (06:00 h) for determination of plasma progesterone as well as oestradiol concentrations on days 6 and 8. Treatment with progesterone was associated with a transient initial decrease (day 1) in both LH pulse frequency and mean LH concentrations after device insertion, but both had returned to pre-treatment levels by day 4. Jersey cows had a greater pulse frequency, but there was no breed difference in mean LH concentrations. Patterns of ovarian follicle growth were affected by progesterone treatment with an increase in diameter of the dominant follicle (DF) identified after treatment initiation. This followed an earlier emergence of a new DF after device insertion. Follicular response to progesterone was dependent on the diameter of the DF present at treatment initiation. Those follicles ≥9 mm were replaced by a new DF during treatment such that the DF observed at the time of device removal was large (≥9 mm) and growing in 13/16 cases. Progesterone was not effective for the induction of an LH surge, ovulation and oestrus in anovulatory cows with a low BCS. However, treatment was associated with synchronous development of a DF so that it was large and growing at the end of the treatment period in most cases. This synchronous development may be due to the transient suppression of LH and the presence of an LH-dependent DF.

Syntheses and Properties of Silicon- and Germanium-Containingα-Amino Acids and Peptides: A Study on C/Si/Ge Bioisosterism

The unnatural silicon-containing α-amino acids (R)- and (S)-H2NCH(CH2SiMe3)COOH [(R)-2 and (S)-2], (R)-H2NCH(CH2SiMe2Ph)COOH [(R)-4], and (R)-H2NCH(CH2SiMe2CHCH2)COOH [(R)-6] as well as the unnatural germanium-containing α-amino acids (R)- and (S)-H2NCH(CH2GeMe3)COOH [(R)-3 and (S)-3] and (R)-H2NCH(CH2GeMe2Ph)COOH [(R)-5] were prepared in three-step syntheses, starting from (R)-3,6-diethoxy-2-isopropyl-2,5-dihydropyrazine [(R)-10]. All amino acids were isolated as enantiomerically pure (≥99% ee) compounds. The (R)- and (S)-enantiomers of β-(trimethylsilyl)alanine [(R)-2 and (S)-2] and β-(trimethylgermyl)alanine [(R)-3 and (S)-3] are sila-analogues and germa-analogues, respectively, of the (S)- and (R)-enantiomers of the nonproteinogenic amino acid β-tert-butylalanine [(S)- and (R)-H2NCH(CH2CMe3)COOH; (S)-1 and (R)-1]. The C/Si/Ge-analogous (l-configurated) amino acids (S)-1, (R)-2, and (R)-3 were treated with (fluoren-9-yl)methyl chloroformate to give the corresponding N-Fmoc derivatives (S)-26, (R)-27, and (R)-28. These N-Fmoc-protected amino acids were used as building blocks for the solid-phase syntheses of the C/Si/Ge-analogous decapeptides 7−9 [Ac-d-Nal1-4-Cl-d-Phe2-d-Pal3-Ser4-Me3El-Ala5-d-Cit6-Leu7-Arg8-Pro9-d-Ala10-NH2 (7, El = C; 8, El = Si; 9, El = Ge)]. The C/Si/Ge analogues 7−9 are derivatives of the GnRH antagonist CetrorelixINN, which bears an (S)-tyrosine residue [instead of the (S)-Me3C-Ala, (R)-Me3Si-Ala, or (R)-Me3Ge-Ala residue] in position 5 of its decapeptide backbone. The decapeptides 7−9 were studied in vitro in receptor binding and functional assays using recombinant cell lines expressing the human GnRH receptor. All compounds behaved as potent GnRH antagonists, the binding affinities and antagonistic potencies of the three C/Si/Ge analogues being quite similar. Compounds 7−9 were also studied for their in vivo activities in the male rat after s.c. administration. They produced both a strong testosterone suppression (single-dose treatment, 1.5 mg/kg) and a strong LH suppression (castrated male rat; single-dose treatment, 0.05 mg/kg). For the silicon- and germanium-containing decapeptides 8 and 9 the testosterone and LH suppression lasted for a significantly longer period of time compared with the effects of the carbon analogue 7.

The LHRH antagonist Cetrorelix: a review

In those clinical situations in which an immediate and profound suppression of gonadotrophins is desired, LHRH agonists have the disadvantage of producing an initial stimulatory effect on hormone secretion. Therefore, the use of GnRH antagonists which cause an immediate and dose-related inhibition of LH and FSH by competitive blockade of the receptors is much more advantageous. One of the most advanced antagonist produced to date is Cetrorelix, a decapeptide which has been shown to be safe and effective in inhibiting LH and sex-steroid secretion in a variety of animal species and in clinical studies as well. Clinical trials in patients suffering from advanced carcinoma of the prostate, benign prostate hyperplasia, and ovarian cancer are currently in progress and have already shown the usefulness of this new treatment modality. In particular, the concept that a complete suppression of sex-steroids may not be necessary in indications such as uterine fibroma, endometriosis and benign prostatic hyperplasia represents a promising novel perspective for treatment of these diseases. Following completion of phase III trials in controlled ovarian stimulation for IVF regimens, Cetrorelix was given marketing approval and, thus, became the first LHRH antagonist available clinically.

A comparative study of high fixed-dose and decremental-dose regimens of gonadotropins in a minidose gonadotropin-releasing hormone agonist flare protocol for poor responders

Selecting the most effective protocol for an IVF procedure is still a major challenge for women known as poor responders to controlled ovarian hyperstimulation (COH). Indeed, using the standard long-term GnRH-agonist (GnRH-a) protocol in these patients requires a large amount of gonadotropins to obtain development of only few follicles. Moreover, the pregnancy rate (PR) remains inappropriately low because of poor oocyte and embryo quality. It was suggested that poor responders may take advantage of the initial endogenous gonadotropin “flare” induced by GnRH-a (1). Moreover, lowering the dose of agonist in short-term GnRH-a protocols may prove effective to decrease the requirement for gonadotropins and to induce an initial gonadotropin release with subsequent LH suppression (2).

Time-related changes in canine luteal regulation: in vivo effects of LH on progesterone and prolactin during pregnancy

The role of LH in luteal function in pregnant dogs was investigated at two different periods during pregnancy: (i) the transitional period from apparent total independence of the corpus luteum to relative hormonal dependence (days 20-35); and (ii) the period of full hormonal dependence (days 35-40). At both periods, LH neutralization, LH inhibition and LH administration studies were conducted. At both periods LH immunoneutralization had no significant effect on the secretion pattern of progesterone or prolactin. GnRH antagonist treatment (Nal-Glu) decreased plasma LH below the detection limit in all treatment periods. Nal-Glu had no effect on prolactin. When GnRH antagonist osmotic pumps were implanted, a transient decrease in plasma progesterone concentrations occurred on days 21-22 but not during the remaining implantation period. When GnRH antagonist was injected, plasma progesterone temporarily decreased (24 h) after the beginning of treatment starting on day 20, but decreased for 5 days when the treatment started on day 35. When purified pig LH was injected i.v. twice a day for 2 consecutive days either from day 30 or from day 40, plasma progesterone concentrations remained constant during treatment. However, on days 40 and 41, an increase in prolactin was observed. These results indicate that LH immunoneutralization may not impair corpus luteum function. In addition, GnRH antagonist induces dose- and time-dependent effects. Only high doses resulted in a decrease in progesterone, the duration of which increased as pregnancy progressed. Continuous GnRH antagonist administration, even when associated with complete LH inhibition, was not associated with detectable effects on progesterone. Finally, LH administration does not stimulate progesterone but may modify prolactin in the last third of pregnancy. Other studies indicated a corpus luteum prolactin dependency. The present study indicates that, in pregnant bitches, LH may not be necessary to sustain progesterone synthesis but that its role may vary in a time-dependent manner.

Inhibitory Effects of Endotoxin on LH Secretion in the Ovariectomized Monkey Are Prevented by Naloxone but Not by an Interleukin-1 Receptor Antagonist

Endotoxin (lipopolysaccharides, LPS), the pathogenic moiety of gram-negative bacteria, is a well-known trigger for the central release of cytokines. The objective of this study is to evaluate the effects of systemic endotoxin administration on LH and cortisol secretion in a non-human primate model and to investigate whether these endocrine effects are mediated by centrally released interleukin-1 (IL-1) using the receptor antagonist to IL-1 (IL-1ra). An additional objective is to investigate whether endogenous opioid peptides mediate these endocrine effects of LPS, using the opiate antagonist naloxone. The experiments were performed in long-term-ovariectomized rhesus monkeys. Blood samples for hormone determination were obtained at 15-min intervals for a period of 8 h, which included a 3-hour baseline period. Since the effective central dose of IL-1ra in the monkey was unknown, in the first experiment we tested the potency of several doses of this antagonist in preventing the effects of centrally administered IL-1α, a cytokine which is known to inhibit LH and stimulate cortisol release. Rhesus monkeys received a 30-min intracerebroventricular infusion of IL-1α (4.2 μg/30 min) alone or together with various doses of IL-1ra (30–180 μg/h i.c.v.). IL-1ra infusion was initiated 1 h before IL-1 and extended over the experimental period. As previously reported, IL-1α induced a significant inhibition of LH, to 36.5 ± 3.3% (mean ± SE) by 5 h as a percentage from the 3-hour baseline. This inhibitory effect was reversed by cotreatment with the 180 µg/h dose of IL-1ra (to 82.5 ± 3.8% by 5 h; NS vs. saline) but not with the lower doses. IL-1 stimulated cortisol release to 165.9 ± 7.7%, but this increase was prevented by IL-1ra (66.6 ± 8.9%; p < 0.05 vs. IL-1, NS vs. saline). In the second experiment, LPS (50 μg) was administered intravenously, alone or in combination with intracerebroventricular IL-1ra infusion. LPS induced a significant decrease in LH secretion (to 57.1 ± 5.2%). These effects were not reversed by intracerebroventricular administration of IL-1ra (52.5 ± 9.6%). Cortisol secretion increased in response to LPS, but this stimulatory effect was not affected by IL-1ra (178.3 ± 13.4 vs. 166.9 ± 5.7%). There were no effects of IL-1ra alone. In experiment 3, we investigated whether the opiate antagonist naloxone reverses the endocrine effects of endotoxin. Both LPS (50 μg) and naloxone (5-mg bolus + 5 mg/h) were infused intravenously. Naloxone was effective in preventing the inhibitory effect of LPS on LH (to 124.6 ± 22.1%, NS vs. saline) but not the increase in cortisol (to 166.7 ± 16.7%; p < 0.05 vs. saline, NS vs. LPS). Naloxone alone has no significant effect on LH or cortisol secretion. These data demonstrate that, in the ovariectomized monkey, a systemic inflammatory/immune- like stress challenge acutely inhibits tonic LH secretion while concomitantly stimulating cortisol release. Although endotoxin is known to affect central cytokine release, these endocrine effects do not require a mediatory role of central IL-1 in the primate. In contrast, endogenous opioid pathways appear to be involved in this process.

Possible involvement of A1 receptors in the inhibition of gonadotropin secretion induced by adenosine in rat hemipituitaries in vitro.

We investigated the participation of A1 or A2 receptors in the gonadotrope and their role in the regulation of LH and FSH secretion in adult rat hemipituitary preparations, using adenosine analogues. A dose-dependent inhibition of LH and FSH secretion was observed after the administration of graded doses of the R-isomer of phenylisopropyladenosine (R-PIA; 1 nM, 10 nM, 100 nM, 1 microM and 10 microM). The effect of R-PIA (10 nM) was blocked by the addition of 8-cyclopentyltheophylline (CPT), a selective A1 adenosine receptor antagonist, at the dose of 1 microM. The addition of an A2 receptor-specific agonist, 5-N-methylcarboxamidoadenosine (MECA), at the doses of 1 nM to 1 microM had no significant effect on LH or FSH secretion, suggesting the absence of this receptor subtype in the gonadotrope. However, a sharp inhibition of the basal secretion of these gonadotropins was observed after the administration of 10 microM MECA. This effect mimicked the inhibition induced by R-PIA, supporting the hypothesis of the presence of A1 receptors in the gonadotrope. R-PIA (1 nM to 1 microM) also inhibited the secretion of LH and FSH induced by phospholipase C (0.5 IU/ml) in a dose-dependent manner. These results suggest the presence of A1 receptors and the absence of A2 receptors in the gonadotrope. It is possible that the inhibition of LH and FSH secretion resulting from the activation of A1 receptors may have occurred independently of the increase in membrane phosphoinositide synthesis.

Normal Suppression of the Reproductive Axis Following Stress in Corticotropin-Releasing Hormone-Deficient Mice*

The hypothalamic neuropeptide CRH has been postulated to inhibit LH secretion by a central action within the brain. To characterize the physiological significance of CRH in stressor-induced inhibition of LH secretion, CRH-deficient and wild-type mice were subjected to restraint or food withdrawal, and plasma LH levels were determined. The proestrus LH surge of female mice was equally suppressed by restraint in both genotypes, and central administration of a CRH antagonist did not alleviate this suppression in either genotype. Male mice of both genotypes also demonstrated suppression of both LH and testosterone secretion following restraint. Furthermore, food withdrawal caused similar suppression of LH secretion in both female and male mice regardless of CRH status. These data demonstrate that CRH is not necessary to inhibit LH secretion following either restraint or food withdrawal and that other molecules are able to suppress LH secretion during the response to stress in the context of CRH deficiency.

Thyroid hormones act primarily within the brain to promote the seasonal inhibition of luteinizing hormone secretion in the ewe.

In the ewe, thyroid hormones are required for the seasonal suppression of GnRH and LH secretion, thereby maintaining an annual rhythm in reproductive activity. The primary site of action of thyroid hormones is unknown; in particular, there is no evidence to distinguish a central from a peripheral action. In this study, we test the hypothesis that thyroid hormones can act directly within the brain to promote GnRH/LH seasonal inhibition. Ovariectomized estradiol-treated ewes were thyroidectomized late in the breeding season to prevent seasonal LH inhibition. T4 was then infused for 3 months, either peripherally or centrally. Neuroendocrine reproductive state was monitored by assaying the LH concentration in biweekly blood samples. Central infusion of low dose T4, which restored a physiological concentration of the hormone in cerebrospinal fluid of these thyroidectomized ewes, promoted the neuroendocrine changes that lead to anestrus. The serum LH concentration in these animals fell at the same time as the seasonal LH decline in euthyroid controls. Neither this same T4 dose infused peripherally nor vehicle infused centrally was effective; LH remained elevated, signifying blockade of the mechanism for anestrus. Our results provide strong evidence that thyroid hormones can act directly within the brain to promote seasonal inhibition of neuroendocrine reproductive function in the ewe.

Disappearance of endogenous luteinizing hormone is prolonged in postmenopausal women.

Pituitary secretion of LH is increased after menopause, but it is not known whether changes in LH clearance also contribute to elevated serum levels. To determine whether the disappearance of endogenous LH is decreased in postmenopausal women (PMW), compared with normal cycling women, GnRH receptor blockade was used to inhibit endogenous secretion of LH and the glycoprotein free alpha-subunit (FAS), and the decline of serum levels was monitored. The NAL-GLU GnRH antagonist ([Ac-D-2Nal1,D-4ClPhe2, D-3Pal3,Arg5,D-4-p-methoxybenzoyl-2-aminobutyric acid6,D-Ala10]GnRH) was administered s.c., at doses of 5, 15, 50, and 150 microg/kg, to 15 euthyroid PMW in 21 studies. Blood was sampled every 10 min, for 4 h before and 8 h after a single sc injection of the GnRH antagonist, followed by hourly samples, ending at 20 h after injection. Results of the maximally suppressive doses (50 and 150 microg/kg) were compared with those of 24 normal cycling women in the early follicular phase and late follicular phase or early luteal phase, and 8 women at the midcycle surge (MCS), who also received these doses of the GnRH antagonist. The best fit curve describing the decay of hormone serum levels after maximal GnRH receptor blockade was determined by nonlinear regression analysis. The elimination of both LH and FAS, after GnRH receptor blockade, exhibited apparent first-order kinetics characterized by a single exponential phase. No differences were seen in percent suppression or half-lives (t1/2) of LH or FAS, between the 50- and 150-microg/kg antagonist doses, in any of the subject populations; and percent suppression of LH was similar across all groups. The t1/2 of LH was prolonged in PMW (139 +/- 35 min, mean +/- est. SD), in comparison with both the MCS (78 +/- 20 min; P < 0.0005) and other cycle stages (57 +/- 28 min; P < 0.0001). However, the disappearance of FAS was not different in PMW, compared with MCS or other cycle stages (t1/2 = 51 +/- 26, 41 +/- 12, and 41 +/- 19 min, respectively). Our conclusions were: 1) Disappearance of endogenous LH after GnRH receptor blockade is significantly prolonged in PMW, compared with the MCS or other cycle stages; 2) The disappearance of FAS is not altered in PMW, suggesting that differences in the disappearance of LH relate to LH microheterogeneity rather than systemic factors.

Effects of Dexamethasone and Dexamethasone plus Naltrexone on Pituitary Response to GnRH and TRH in Normal Women

The hypothesis that glucocorticoids have a direct central inhibitory effect on the reproductive axis is sutained by the identification of glucocorticoid receptors on GnRH-secreting neurons and gonadotropic pituitary cells. It has been proposed that glucocorticoids and opioids interact centrally in the regulation of the GnRH-LH axis. The inhibitory effect of glucocorticoids may manifest not only directly through the hormone-receptor link, but also indirectly through an increase in opioid tone. The aim of this study was to evaluate the role of glucocorticoids and glucocorticoids combined with an opioid antagonist, in the regulation of basal and GnRH- and TRH-stimulated secretion of LH, FSH and Prl in 7 women with normal menstrual cycles. Blood samples were obtained every 10 min for an hour. GnRH (50 μg) and TRH (200 μg) were administered and blood sampling was continued every 15 min for 2 h (day 1). At 5 a.m. the next day, naltrexone (50 mg) was given and at 8 a.m. the GnRH-TRH test was repeated (day 2). At 5 a.m. on day 3, the patients took 2 mg oral dexamethasone and the test was repeated. At 5 a.m. on day 4, the patients took naltrexone and dexamethasone and at 8 a.m. the GnRH-TRH test was repeated. Administration of naltrexone did not cause significant changes in basal concentrations of LH and FSH and their response to GnRH. The area under the curve of the LH response to GnRH on day 3 was significantly less than on days 1, 2 and 4. Administration of naltrexone (day 2) did not cause any significant increase in basal and TRH-stimulated levels of Prl with respect to day 1. On day 3, dexamethasone caused a reduced response of Prl to TRH. Pretreatment with naltrexone (day 4) prevented this reduction. These results suggest that suppression of the response of LH to GnRH induced by dexamethasone may be partly mediated by endogenous opioids. Dexamethasone led to a reduction in the response of Prl to TRH, and naltrexone blocked this suppression. Hence the suppression of Prl and LH by dexamethasone must be partly mediated by endogenous opioids, which must therefore inhibit pituitary secretion of Prl.

Gonadotropin stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) messenger ribonucleic acid in the rat ovary and the role of PACAP as a follicle survival factor.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide with considerable homology to vasoactive intestinal peptide and GH-releasing hormone, exists in two biologically active forms, PACAP-38 and -27. The presence of PACAP in the ovary has been demonstrated, where it stimulates steroidogenesis and cAMP accumulation in cultured granulosa cells. In the present study, gonadotropin regulation of PACAP gene expression was examined in PMSG/human (h)CG-treated immature rat ovaries and cultured preovulatory follicles. Northern blot analysis of ovaries obtained from PMSG/hCG-treated immature animals revealed the transient induction of PACAP transcripts by hCG, reaching a maximum at 6 h. The major cell types expressing PACAP messenger RNA were granulosa cells of preovulatory follicles and some theca/interstitial cells. In preovulatory follicles cultured in serum-free medium, PACAP transcripts were transiently induced by LH and FSH, reaching a maximum 6-9 h after stimulation in granulosa cells but not in theca cells. Treatment with cycloheximide or alpha-amanitin abolished LH-induced PACAP transcripts, indicating that new protein synthesis and transcription are necessary. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-induced PACAP messenger RNA, and forskolin mimicked the LH action, implying the role of adenylate cyclase activation. In contrast, treatment with chelerythrine, an inhibitor of protein kinase C, and 2-O-tetradecanol-phorbol-13-acetate had no effect. We further tested the role of PACAP in follicle apoptosis using apoptotic DNA fragmentation analysis. Treatment with PACAP-38 suppressed follicle apoptosis in a dose-dependent manner. Moreover, the LH suppression of follicle apoptosis was partially blocked by cotreatment with PACAP-38 antagonist, indicating mediation by endogenous PACAP-38. These results suggest that PACAP, transiently induced by the gonadotropin surge, could be a local regulator of a number of events and may act as a follicle survival factor during the periovulatory period.

Suppression of spermatogenesis in man induced by Nal-Glu gonadotropin releasing hormone antagonist and testosterone enanthate (TE) is maintained by TE alone.

GnRH antagonists plus testosterone (T) suppress LH and FSH levels and inhibit spermatogenesis to azoospermia or severe oligozoospermia. High-dose T treatment alone has been shown to be an effective male contraceptive (contraceptive efficacy rate of 1.4 per 100 person yr). Combined GnRH antagonist and T induces azoospermia more rapidly and at a higher incidence than T alone; this combination has therefore been proposed as a prototype male contraceptive. However, because GnRH antagonists are expensive to synthesize and difficult to deliver, it would be desirable to rapidly suppress sperm counts to low levels with GnRH antagonist plus T and maintain azoospermia or severe oligozoospermia with T alone. In this study, 15 healthy men (age 21-41 yr) with normal semen analyses were treated with T enanthate (TE) 100 mg im/week plus 10 mg Nal-Glu GnRH antagonist sc daily for 12 weeks to induce azoospermia or severe oligozoospermia. At 12-16 weeks, 10 of 15 subjects had zero sperm counts, and 14 of 15 had sperm counts less than 3 x 10(6)/mL. The 14 who were suppressed on combined treatment were maintained on TE alone (100 mg/week im) for an additional 20 weeks. Thirteen of 14 subjects in the TE alone phase had sperm counts maintained at less than 3 x 10(6)/mL for 20 weeks. Ten remained persistently azoospermic or had sperm concentration of 0.1 x 10(6)/mL once during maintenance. Mean LH and FSH levels in the subjects were suppressed to 0.4+/-0.2 IU/L and 0.5+/-0.2 IU/L in the induction phase, which was maintained in the maintenance phase. The 1 subject who failed to suppress sperm counts during induction had serum LH and FSH reduced to 0.3 and 0.5 IU/L, respectively. The subject who failed to maintenance had LH and FSH suppressed to 1.0 and 0.2 IU/L, respectively, during the induction phase but these rose to 1.6 and 2.1 IU/L, respectively, during maintenance. Failure to suppress or maintain low sperm counts may be related to incomplete suppression of serum LH and FSH levels. We conclude that sperm counts suppressed with GnRH antagonist plus T can be maintained with relatively low dose TE treatment alone. This concept should be explored further in the development of effective, safe, and affordable hormonal male contraceptives.

Pulsatile luteinizing hormone secretion in the ovariectomized, thyroidectomized red deer hind following treatment with dopaminergic and opioidergic agonists and antagonists.

Two experiments were conducted to determine whether dopaminergic or opioidergic pathways are modulated by thyroid gland secretions for seasonal suppression of LH secretion in red deer hinds. Ovariectomized (n = 5) or ovariectomized and thyroidectomized (n = 4) hinds, treated with estradiol implants, received the dopamine agonist bromocriptine or the antagonist sulpiride during pulse bleeds in July (breeding season) and October (nonbreeding season). Comparison of July and October mean plasma LH concentration (3.5 +/- 1.3, 0.7 +/- 0.1 ng/ml, respectively), pulse frequency (1.9 +/- 0.4, 0.7 +/- 0.2 pulses/4 h), and pulse amplitude (1.3 +/- 0.5, 0.7 +/- 0. 02 ng/ml) showed lower (p < 0.05) levels in October, and these levels were not significantly affected by thyroidectomy or drug treatment. In the absence of estradiol implants, the hinds received bromocriptine or morphine during the breeding season (July) and their antagonists, sulpiride or naloxone, respectively, in the nonbreeding season (November). In euthyroid hinds there was a seasonal decrease (p < 0.05) in mean plasma LH concentration, pulse frequency, and pulse amplitude, which did not occur in thyroidectomized hinds. There were no effects of drug treatment on LH concentration except for a small increase following sulpiride in November. Plasma prolactin concentration was significantly increased by antagonists and decreased by agonists on most occasions. We conclude that in red deer hinds, seasonal regulation of LH secretion does not involve dopamine or endogenous opioids and the thyroid gland is required specifically for LH suppression in the absence of estradiol.

The effect of season and melatonin on GnRH-induced LH secretion in oestradiol-treated orchidectomized sheep.

The biphasic effect of oestradiol (E2) on gonadotrope responsiveness is clearly evident in orchidectomized sheep (wethers) receiving E2 and hourly pulses of GnRH. We hypothesized that the duration of E2-induced reduction in gonadotrope responsiveness differed between the breeding (November) and anoestrous (May) seasons in sheep. To test this hypothesis wethers (n = 6/group) were infused (i.v.) with E2 (2 micrograms/50 kg per h) and received hourly pulses of GnRH (200 ng/50 kg per pulse) or saline in May or November. The pattern of LH secretion during the 72 h infusion period was determined. Serum concentrations of LH did not differ with season in control wethers receiving vehicle alone. Similarly, continuous infusion of E2 resulted in a 3-fold reduction in serum LH, irrespective of season. This E2-induced suppression of serum LH was reversed by concurrent episodic delivery of GnRH. The interval between initiation of infusion and return of pretreatment concentrations of LH was taken as a measure of the duration of E2-induced suppression of gonadotrope responsiveness. The duration of this E2-dependent response varied with season, with suppression of gonadotrope responsiveness more prolonged (P < 0.05) in May (36.7 +/- 2.9 h) than in November (14.3 +/- 1.1 h). In a companion study we examined the effect of melatonin on the duration of E2-induced suppression of gonadotrope responsiveness. Wethers received blank or melatonin-containing implants in March. Sixty days after implant insertion (mid-May) wethers received E2 (2 micrograms/50 kg per h) and hourly pulses of GnRH (200 ng/50 kg per pulse) or saline for 72 h. Continuous delivery of E2 alone resulted in a 3-fold decrease in serum concentrations of LH in both control and melatonin-treated wethers. The duration of E2-induced suppression of gonadotrope responsiveness in animals receiving E2 and GnRH was extended (P < 0.05) in wethers with blank implants (48.0 +/- 0.7 h), relative to the duration of suppression in melatonin-treated wethers (14.5 +/- 1.0 h). Taken together these data indicate that E2-induced suppression of gonadotrope responsiveness is more extended during the anoestrous season. However, this seasonal effect can be reversed by continuous administration of melatonin.