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Five women with amenorrhea of suprapituitary origin 'were given intravenous injections of 10 µg LH-RH every 90 minutes for 4 days by means of a portable infusion pump. Immediately before and after this, the LH and FSH responses to a test dose of 100 µg LH-RH were measured.Four days after discontinuation of the treatment, so that LH and FSH could be measured, blood was sampled every 10 minutes for a period of 6 hours, during which 20 µg LH-RH was injected intravenously every hour. Finally, a test dose of 100 µg LH-RH was given. The whole procedure was repeated at least 6 weeks later, but this time hourly injections of 100 µg LH-RH were given 4 days after discontinuation of the pulsatile LH-RH treatment.Four days after the pulsatile LH-RH treatment was stopped, increased LH and FSH responses to LH-RH were observed. These could be reduced by 6 injections, given hourly, of either 20 or 100 µg LH-RH. Although the totally released amount of both LH and FSH did not differ between the two treatment regimens irrespective of the LH-RH dose used, the response of both gonadotropins to the LH-RH test dose after the hourly 100 µg LH-RH injections was significantly lower. This indicated that desensitization can be attributed, at least in part, to a lower responsiveness of LH and FSH to LH-RH when pulsatile LH-RH is given. Low responses during treatment with pulsatile LH-RH could not be related to higher concentrations of plasma estradiol.We conclude that women with amenorrhea of suprapituitary origin who are treated with pulsatile LH-RH have a low state of responsiveness to LH-RH, which can be caused by the presence of the LH-RH and might be attributed in part to desensitization by LH-RH. Removal of the LH-RH results in an enhancement of the responsiveness, as the pituitary gland might have recovered from this desensitization.

The hypothesis that LH-RH induces LH release partly through a protein synthesis dependent step (protein factor) was further investigated using two different experimental designs.First, during incubation of pituitary glands of intact dioestrous female rats with a maximally active concentration of LH-RH, the inhibitor of protein synthesis cycloheximide was added at various times after the beginning of the incubation. The results show that it takes a relatively long time, i.e. more than 1 h of exposure to LH-RH before the amount of the protein factor has increased sufficiently to allow a maximal LH secretion.Secondly, LH-RH was injected iv after which the protein factor was assayed by incubating the pituitary glands with a maximally active concentration of LH-RH in the presence of cycloheximide and measuring LH release in vitro. It was found that 1 h after the injection sufficient protein factor was present to permit an elevated response to LH-RH. This response could be suppressed by injecting cycloheximide prior to LH-RH. When the interval between injection of LH-RH and beginning of the incubation was increased to 2 h, LH release in vitrodecreased again. However, ovariectomy immediately before LH-RH injection resulted in maintenance of the elevated response to LH-RH in vitro, indicating a role of the ovaries in this phenomenon.

Continuous exposure of hemi-pituitary glands from intact female rats to LH releasing hormone (LH-RH) in vitrodisplayed three phases in the pattern of LH release: during the first hour release of LH was low (first phase response), then it increased to a higher level during the second hour and remained constant during the next 2 h (second phase response), after which there was a refractoriness of LH release (third phase response). The initial phase response of pituitary glands from intact rats was blocked by EGTA (a Ca2+chelator) but there was a small but significant increase in the rate of LH release during the second phase response. This increase could be prevented by inhibition of protein synthesis by cycloheximide. Cycloheximide and EGTA did not affect basal release of LH by glands from intact rats, neither did EGTA affect the high basal release of LH by glands from ovariectomized rats. However, the LH-RH-induced release of LH from pituitary glands of ovariectomized rats, which did not show the initial phase of low LH release, was completely suppressed by EGTA throughout a 4-h incubation period.The pattern of LH release stimulated by the combination of N6-monobutyryl cyclic AMP and theophylline (mbcAMP/theophylline) showed an initial phase of low LH release lasting 4 h after which it increased. The magnitude of the effect was small compared with the action of LH-RH. As it did with LH-RH, EGTA completely blocked the initial response, but allowed a small increase in the rate of LH release thereafter; this increase could also be blocked by inhibition of protein synthesis.Addition of EGTA to media during pretreatment of pituitary glands from intact rats with either LH-RH or mbcAMP/theophylline did not impair the facilitatory effect of these secretagogues on the responsiveness of the glands to subsequent exposure to LH-RH and cycloheximide and normal Ca2+levels. The restoration of Ca2+levels after withdrawal neither affected basal nor LH-RH-induced release of LH.Exclusion of Ca2+from the media during a 6-h incubation of pituitary glands from intact rats with LH-RH prevented the glands from becoming refractory to subsequent stimulation by LH-RH, which occurs when normal Ca2+concentrations are present.The results suggested that extracellular Ca2+is obligatory for LH release and the induction of refractoriness by LH-RH. In contrast, that part of the action of LH-RH which is cyclic AMP-mediated and protein synthesis-dependent is not affected by withdrawal of extracellular Ca2+.

Summary.The implantation of Silastic capsules containing androstenedione (release rate 63·2 ± 4·4 μg/24 h) into 4-day cyclic rats resulted in a prolongation of the cycle (P< 0·001), most rats showing 5-day cycles after the first, largely unaffected cycle. There was a reduction in ovulation rate (P< 0·01) and lower serum LH levels on the morning of oestrus (P< 0·01) but serum FSH levels were unaffected.

Pulsatile release of LHRH and short-term pituitary desensitization to LHRH in the rat are believed to be necessary for the maintenance of LH pulsatility. In contrast, FSH release is partly induced by LHRH release and is partly LHRH-independent. This LHRH-independent release of FSH is subject to inhibitory feedback control by ovarian proteins (probably inhibin), and may obscure an LHRH-induced shortterm loss of pituitary FSH responsiveness to LHRH. The object of this study was to establish whether short-term pituitary desensitization to single doses of LHRH results not only in a loss of LH response, but also of FSH response. Ovariectomized rats were used to eliminate the influence of steroid feedback.A group of ovariectomized rats was pretreated with steroid-free bovine follicular fluid (bFF) to suppress LHRH-independent FSH release, and phenobarbital to suppress LHRH-dependent FSH release respectively, 7 and 1 h before administration of LHRH. Another group received phenobarbital only. The animals were injected sequentially with either low or high doses of LHRH (1·25 or 10 ng/100 g body weight at times 0 and at 80, 120 or 180 min, and 6·25 or 50 ng/100 g at 60 min). Blood was taken for FSH measurements before and 5 and 10 min after each injection. Rats pretreated with bFF and phenobarbital showed an acute FSH response related to the dose of injected LHRH. No dose–response curve was seen in animals which had only been pretreated with phenobarbital. A significantly attenuated FSH response to LHRH injections of 1·25 or 10 ng/100 g, given 20 min after an injection of 6·25 or 50 ng LHRH/100 g body weight respectively, was observed in animals pretreated with a combination of phenobarbital and bFF, but not in those treated with phenobarbital alone.The present results confirm that FSH release is under dual control by LHRH and ovarian secretory proteins. When both LHRH-dependent FSH release and the LHRH-independent FSH release are suppressed, short-term desensitization of FSH release to exogenous LHRH can be demonstrated. It is concluded that the phenomenon of short-term pituitary desensitization to LHRH and inhibitory ovarian proteins may both play a role in the regulation of FSH release.Journal of Endocrinology(1989) 123,221–226

When pituitary glands from intact female rats are incubated with LHRH, the resulting LH release shows a biphasic pattern: an initial low rate of LH release (lag phase) is followed by a high rate. When pituitary glands from long-term ovariectomized rats are incubated, the rate of LH release is high throughout stimulation with LHRH. The disappearance of the lag phase might be due to increased LHRH release after ovariectomy and/or the disappearance of ovarian factors.To distinguish between these possibilities, pituitary glands which had been exposed to endogenous LHRH (pituitary glands in situ) or which had been unexposed to endogenous LHRH (pituitary glands transplanted under the kidney capsule) were incubated in the presence or absence of LHRH.Biphasic LH secretion patterns were observed during incubation with LHRH with the animal's own pituitary gland and with the transplanted pituitary gland from intact, but not from ovariectomized rats. Thus the disappearance of the lag phase after ovariectomy results from the absence of ovarian secretory products, rather than from increased release of LHRH.Journal of Endocrinology(1989) 123,41–45

Abstract. The effect of pulsatile GnRH administration on the levels of LH and FSH was investigated in rats that had been ovariectomized 2 weeks earlier. Also the asynchronous occurrence of endogenous and GnRH-induced LH and FSH pulses was analysed. A small pulse dose of GnRH (1.25 ng/100 g) was given iv at a frequency of once every 60 min or once every 120 min during 24 h. A larger dose of 5 ng/100 g was given once every 60 or 120 min during either 24 h or 96 h. Blood was sampled arterially every 5 min around the two first and last GnRH injections and LH and FSH were measured. Only the treatment with the larger GnRH pulse dose resulted in a change of LH and FSH plasma levels. LH levels declined under all circumstances, whereas FSH was found to be increased temporarily after 24 h of treatment. The pituitary LH response to pulses of GnRH (5 ng/100 g) decreased irrespective of the frequency or duration with which GnRH was administered. There was a marked asynchronicity between LH and FSH pulses and almost every injection of GnRH (5 ng/100 g) resulted in clear LH pulses but not in FSH pulses.

Abstract. Otherwise untreated adult ovariectomized rats were given pulses of GnRH (5 ng/100 g body weight iv) once every 60 or 120 min for 24 or 96 h. On the first and last day of the experiment plasma LH was estimated during the administration of GnRH pulses. Endogenous LH pulses between exogenously generated LH pulses were observed in nearly all animals on both the first and the last day, without any change in nadir and amplitude values. Shortly after an injection of GnRH, the spontaneous LH pulses were fewer than expected. The number of these pulses, however, increased again with time after the injections. The response to exogenous GnRH was reduced on the last day of the experiment. However, not all GnRH injections led to LH pulses. Most injections which did not result in an LH pulse appeared to be given within 15 min after a preceding endogenous LH pulse. The results obtained are in agreement with the hypothesis of an acute short-lasting desensitization of the pituitary gland caused by exogenous as well as endogenous pulses of GnRH.

Abstract. Ovariectomized rats were injected iv with an antiserum against LRH or normal rabbit serum. AntiLRH caused a decrease of plasma LH and FSH. After 24 or 48 h, the rats were decapitated and the pituitary glands incubated in the presence of an analogue of LRH which reacts minimally with anti-LRH (Buserelin). Pretreatment with anti-LRH caused an increased response of pituitary LH release to Buserelin. Similar results were obtained with regard to FSH. In this case, however, basal release of FSH was lowered by pre-treatment with antiLRH. Pituitary LH and FSH contents were not affected by anti-LRH, but synthesis of LH and FSH in vitro was smaller than in control glands obtained from rats pretreated with normal rabbit serum.

Abstract. Gonadotropin secretion was inhibited in immature male and female rats by scadministration of the GnRH-antagonist ORG30276 (GnRH-A) on days 6, 9, 12 and 15. In GnRH-A-treated females this resulted in suppression of the temporarily increased plasma LH and FSH levels, which normally occur in prepubertal female rats. Ovarian weight was decreased. Although vaginal opening in GnRH-A-treated rats occurred earlier, the age of 1st estrus and the number of ova shed at first ovulation were not affected. Fertility at 4 months of age was normal. After initial suppression of gonadotropin levels, the FSH levels in GnRH-A-treated males, however, sharply increased to about twice the control levels. Plasma FSH remained elevated at least until 4 months of age. The LH levels at adult age were not affected by antagonist treatment and neither were testosterone levels. Testicular weight was decreased by GnRH-A from about 50% on day 15 to about 30% at 4 months of age. Pubertal development as measured by balano-preputial separation was delayed by about 7–10 days. At 4 months of age fertility was decreased. Thus, suppression of early gonadotropin secretion by GnRH-A treatment had dramatic effects on subsequent pubertal development in the male, but not in the female rat.

Abstract. The effect of a single scinjection of an LRH antagonist ((Ac - D - p - Cl - Phe1,2,D-Trp3,D-Phe6,D-Ala10)-LRH, Org 30093) into OVX rats on FSH release 24 h later was studied. Plasma FSH was decreased but pituitary FSH content was not changed. Incubation of the pituitary glands during 4 h resulted in a decreased basal release. FSH release induced by a low concentration of LRH (1 ng/ml) was depressed but that of a high concentration (10000 ng/ml) was augmented in comparison to FSH release induced in control glands. However, pretreatment with the antagonist had no specific effect on FSH release in vitro induced by high K+or high K+plus mbcAMP and theophylline, indicating that the changes of pituitary responsiveness to LRH are not caused by those parts of the secretory mechanism which are stimulated by these secretagogues. Moreover, it is concluded that the changes of pituitary LH release induced by administration of an LRH antagonist also concern FSH.

The bioassay for LH-like hormones, based on the amount of testosterone produced in vitroby collagenase-dispersed rat Leydig cells in response to the hormone to be tested, was used to see whether a chorionic gonadotrophin (CG)-like hormone could be detected in the serum of pregnant rats. The curve of the serum “CG” concentration (i.e. in vitrotestosterone produced) during the course of pregnancy was different from that of serum LH, determined by RIA, but was very similar to that of serum progesterone. This similarity proved to be due entirely to the conversion of progesterone to testosterone by the Leydig cells, since known amounts of progesterone (10-200 ng) dissolved in hypophysectomized rats'serum were converted quantitatively to testosterone when added to the cells under the same conditions used to test the pregnancy serum. The bioassay may thus be used as a specific measure of LH-like hormones only under conditions in which it is certain that steroids which can be converted to testosterone are either removed, or are present in amounts too small to have a significant effect on the values obtained.

Ovariectomized (OVX) rats were treated for 24 h with pulses of LRH (1.25 ng/100 g b.w. every 20 min) or of saline. Moreover, the animals received 3 sc injections of oestradiol benzoate (EB) (50 μg) or oil during this period. At the end of this period all animals received phenobarbital (80 mg/kg ip) and the animals were given pulses of LRH (once every 20 min) containing 1.25, 2.5, 5.0 or 10 ng for 3 h. Pulsatile administration of 1.25 ng pulses for 24 h did not lead to pituitary insensitivity. Treatment with EB caused the development of an increased response to LRH (positive effect). When during the 24–27 h period 5.0 or 10.0 ng pulses of LRH were given, the positive response was the same whether LRH had been administered during the 0–24 h period or not. However, the response to 2.5 ng pulses of LRH was halved when the animals had been pre-treated with LRH. It is concluded that the presence of LRH can inhibit partly the development of the positive pituitary response to LRH as caused by EB.When the dose of LRH was kept at 1.25 ng/pulse no clear positive effect developed. Chronic administration of phenobarbital for 24 h caused pituitary insensitivity, presumably through a direct toxic effect.

The involvement of cyclic AMP in the action of LH releasing hormone (LH-RH) on LH secretion was studied by incubating pituitary glands from adult female rats on day 2 of dioestrus with 1 mm-N6-monobutyryl cyclic AMP (mbcAMP) and 10 mm-theophylline for periods of up to 10 h. This treatment induced a pattern of LH release similar to that observed in the presence of a low concentration of LH-RH (0·1 ng LH-RH/ml), i.e. an initial 4 h period during which the release of LH was minimal was followed subsequently by an increased rate of release. In this system inhibition of protein synthesis by cycloheximide (25 μg/ml) did not impair the initial response of the pituitary tissue but the increase in the rate of LH release during the second phase of the response was blocked.Preincubation with mbcAMP and theophylline increased the responsiveness of the pituitary tissue to LH-RH. This action could be prevented by including cycloheximide during the preincubation period, whereas addition of this drug during the incubation with LH-RH no longer impaired the increased responsiveness. The size of the sensitizing action of mbcAMP and theophylline mediated through the induction of protein synthesis was comparable with that of a high concentration of LH-RH. From the absence of a significant change in total LH during the preincubation period, it was concluded that the increased responsiveness was not the result of newly synthesized LH.The present results suggest a role or roles for cyclic AMP in the secretion of LH induced by LH-RH. Besides an effect on the formation of a factor related to the synthesis of protein, other than LH which has a permissive role in the acute release of LH, cyclic AMP might also be concerned in the secretion process through a pathway which does not involve synthesis of protein.

The refractoriness of LH release by pituitary glands from intact female rats was studied during stimulation by luteinizing hormone releasing hormone (LH-RH), monobutyryl cyclic AMP+theophylline or potassium in vitro. Various concentrations of LH-RH (0·1, 0·3 and 10 ng/ml) all caused refractoriness within 24 h. Subsequent exposure to a supramaximally active concentration of LH-RH for 6 h also resulted in a depressed response; the degree of inhibition depended on the concentration of LH-RH to which the glands had been exposed previously. Glands made refractory to LH-RH also showed a depressed response to monobutyryl cyclic AMP+theophylline, although these agents by themselves were unable to induce refractoriness. Incubation in medium containing a high concentration of potassium also resulted in the release of LH, which in all respects was similar to that caused by LH-RH. Glands made refractory to LH-RH showed a decreased response to potassium and, conversely, the release of LH in response to LH-RH was reduced after exposure of glands to potassium.It is concluded that the LH releasing activity of LH-RH, which is mimicked by potassium, deteriorates during continuous exposure to the secretagogue.

The aim of the study was to test the hypothesis that in serial determinations of concentrations of LH and FSH involving blood samples taken every minute, the observed pulses of LH and FSH which last less than 3–4 min might not be a physiological phenomenon but part of the 'noise' of the radioimmunoassay or blood-sampling technique.Blood was sampled every minute for a period of 90 min in six men. During the first 45 min, blood was sampled by means of vacuum tubes only. During the second 45 min, sampling took place with a syringe via a rubber stopper, either using a tourniquet (n= 3) or flushing the cannula with heparinized saline.Three criteria were used to identify variations in the patterns of LH and FSH as true hormonal changes. First, a threshold was used which had to be exceeded by the difference between nadir and maximum values before a pulse could be identified. An average of approximately six pulses per 90 min was found in both the LH and FSH series. The majority of these pulses lasted less than 3–4 min. In two subjects, larger LH pulses of longer duration were measured. Secondly, differences between duplicate measurements of nadir and/or maximum values of more than one-third of the amplitude of a pulse were considered unacceptable. This involved about 75% of the pulses. Thirdly, the reproducibility of the hormone variations was estimated. In one subject, concentrations of LH were measured four times in four separate assays. Measurement of FSH concentrations in this subject and of LH and FSH in the samples from the other five subjects were repeated once again, but only in those parts of the series of samples which had shown hormone variations beyond the threshold composed of acceptable duplicate measurements. Only the larger variations of longer duration, as found earlier in two of the LH series, were reproducible.The different blood-sampling techniques used had no significant influence on the frequency of pulses.It was concluded that a rapid, small amplitude, pulsatile pattern of release of LH and FSH is probably not present in men or is obscured by limitations of current techniques of radioimmunoassay.J. Endocr.(1987) 114,153–160

When pituitary glands from intact female, but not from ovariectomized rats, are incubated for 8 h in medium TC199 without further additives, FSH is synthesized. This LHRH-independent (or autonomous) FSH synthesis is prevented when bovine follicular fluid (bFF) is added to the incubation medium.Results from preliminary experiments, however, indicate no clear autonomous FSH synthesis after long-term absence of LHRH. To investigate the regulatory mechanisms involved in autonomous FSH synthesis and release, pituitary glands (exposed to endogenous LHRH) and pituitary grafts (not exposed to endogenous LHRH) from intact and ovariectomized rats were incubated for 8 h in medium TC199. Total FSH content (FSH released plus FSH remaining in the tissue) was compared with that in non-incubated glands or grafts, giving an indication of FSH synthesis. In addition, some of the animals were given LHRH pulses for 40 h before incubation. When pituitary tissue was taken from intact female rats, FSH synthesis occurred in the animals' own glands and in grafts from LHRH-pretreated rats. No FSH synthesis was seen in ovariectomized rats with or without pretreatment with bFF and/or LHRH. However, when ovariectomized rats had been pretreated with oestrogen, FSH synthesis was measured in vitroafter pulsatile LHRH treatment in vivo.The results indicate that autonomous FSH synthesis in vitrois dependent upon previous (in vivo) exposure of the glands to both oestrogen and LHRH.Journal of Endocrinology(1991) 129,27–33

Abstract. In the ovariectomized (OVX) rat, the plasma LH response was measured to a pulse of LRH (1.25 or 5 ng/100 g body weight, ia) given before and 1 h after ip administration of phenobarbital (80 mg/kg body weight). The LH response to the LRH pulses was increased 1 h after phenobarbital.In a second experiment, the pituitary LH content of OVX rats was measured 1 h after administration of phenobarbital or saline. No difference in pituitary LH content was found.It is concluded that in the OVX rat, phenobarbital increases the response to a pulse of LRH, presumably by suppressing endogenous pulsatile LRH. This, together with results of earlier experiments, further supports the hypothesis that under conditions where endogenous pulsatile LRH is present, there is always a certain degree of pituitary desensitization or refractoriness and that the removal of this endogenous LRH leads to recovery of pituitary sensitivity to LRH.

Castration of male rats decreases both pituitary and serum TSH-levels. Administration of testosterone propionate increases serum TSH-levels in castrated males, but its effect on pituitary TSH-content appears to be complex: whereas treatment with physiological amounts of testosterone prevents the decrease induced by castration, administration of high doses of testosterone results in low pituitary TSH-contents not unlike those seen in untreated castrated rats. Testosterone administered to thyroxine-maintained thyroidectomized rats had the same effects as mentioned above, but if thyroxine treatment was omitted, no effect of testosterone on pituitary and serum TSH-levels could be observed. It is thought that testosterone interferes with the effect of thyroid hormone on the secretion of TSH.