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J. DE KONING, J. A. M. J. VAN DIETEN and G. P. VAN REES

Leiden University Medical Centre, Department of Pharmacology, Wassenaarseweg 72, 2333-AL Leiden, The Netherlands

(Received 17 November 1977)

The mechanism by which oestrogen can augment the pituitary response to luteinizing hormone releasing hormone (LH-RH) is unknown. A number of studies have suggested that at least part of the action of LH-RH is mediated by cyclic AMP (Ratner, 1970; Borgeat, Chavancy, Dupont, Labrie, Arimura & Schally, 1972; Makino, 1973; Beaulieu, Labrie, Coy, Coy & Schally, 1975). In preliminary experiments we found that the combination of 1 mm-N 6-monobutyryl cyclic AMP plus 10 mm-theophylline showed maximum activity in causing the release of luteinizing hormone (LH) from the pituitary glands of intact dioestrous rats in vitro, although it only mimicked the action of a relatively low concentration of LH-RH (0·1 ng/ml). We decided to investigate whether the release of LH induced by this combination of monobutyryl cyclic AMP and theophylline could also be augmented by

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J. A. M. J. van Dieten, J. de Koning and G. P. van Rees

ABSTRACT

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

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J. A. M. J. van Dieten, J. de Koning and G. P. van Rees

ABSTRACT

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 vitro after pulsatile LHRH treatment in vivo.

The results indicate that autonomous FSH synthesis in vitro is dependent upon previous (in vivo) exposure of the glands to both oestrogen and LHRH.

Journal of Endocrinology (1991) 129, 27–33

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J. DE KONING, J. A. M. J. VAN DIETEN and G. P. VAN REES

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.

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J. de Koning, A. M. I. Tijssen and G. P. van Rees

ABSTRACT

The effects of discontinuation and restoration of ovarian influences on the pituitary LH response to LHRH in vitro were investigated.

When female rat pituitary glands taken on day 2 of dioestrus were incubated with LHRH the release of LH was low during the first hour (lag phase response) and afterwards a progressive, protein synthesis-dependent increase took place (second phase response), this being the self-priming action of LHRH.

Short-term discontinuation (less than 1 day) of ovarian influences on the rat pituitary gland in vivo (ovariectomy) or in vitro (incubation in medium only) resulted in an increased LHRH-induced LH response during the lag phase.

The biphasic LH response or the self-priming action of LHRH disappeared completely after long-term discontinuation of ovarian influences on the pituitary gland, LH release being at its maximum from the start of the incubation. The biphasic response was reinstated when ovaries were implanted under the kidney capsules of ovariectomized rats. Auto-implantation of an ovary into the spleen immediately after bilateral ovariectomy did not, however, prevent the disappearance of the LHRH self-priming action. Ovarian activity responsible for the presence of the low LH response during the lag phase was thus effectively removed by the liver, but inhibin-like activity suppressing serum FSH levels remained present. Silicone elastomer implants (s.c.) containing oestradiol-17β, implanted for 4 weeks, did not reverse the loss of the biphasic LH response to LHRH.

It is concluded that liver-labile factors released by the ovaries keep the pituitary gland in a state of low responsiveness to LHRH. By giving a sufficiently high LHRH stimulus this inhibitory effect is neutralized and transition to a highly responsive state can be achieved. The ovarian factor(s) is not identical to inhibin or oestradiol-17β.

J. Endocr. (1987) 112, 265–273

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J. de Koning, A. M. I. Tijssen and G. P. van Rees

ABSTRACT

Pituitary glands taken from intact rats on day 2 of dioestrus and incubated with LHRH show a biphasic pattern of LH and FSH release. Initially the release of the gonadotrophins is low (first-phase or lag-phase response), but increases during further incubation with LHRH (second-phase or primed-state response).

Removal of the influence of an unidentified ovarian factor either by ovariectomy or prolonged incubation in medium only leads to an increased (lag-phase) response to LHRH. The development of the increased response after prolonged incubation was prevented by the addition of cycloheximide to the media, implicating that this process is dependent upon the synthesis of protein. Steroid-free material (bovine follicular fluid or rat ovarian extracts) prevented the development of this process. In addition, it was shown that steroid-free rat ovarian extracts were also able to induce the development of a lag phase in pituitary glands from ovariectomized rats. Finally, it was found that steroid-free ovarian extracts reversed the self-priming effect of LHRH.

The biological activity which reduced the responsiveness of the pituitary gland towards stimulation by LHRH was eliminated after the use of protein-denaturating techniques such as increased temperature or addition of methanol. The presence of this activity in ovaries, did not vary during the oestrous cycle, contrary to inhibin-like activity. Hence the ovarian factor responsible for the low lag-phase response is a protein which is probably not identical to inhibin.

It is concluded that a non-steroidal ovarian factor reduces the responsiveness of the anterior pituitary gland to LHRH. This reduced responsiveness can be reversed by LHRH, which is generally recognized as the self-priming effect of LHRH.

Journal of Endocrinology (1989) 120, 439–447

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C. B. Lambalk, G. P. van Rees, J. Schoemaker, J. de Koning and J. A. M. J. van Dieten

ABSTRACT

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

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J. de Koning, A. M. I. Tijssen, J. A. M. J. van Dieten and G. P. van Rees

Continuous exposure of hemi-pituitary glands from intact female rats to LH releasing hormone (LH-RH) in vitro displayed 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+.

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J. DE KONING, J. A. M. J. VAN DIETEN, A. M. I. TIJSSEN and G. P. VAN REES

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-N 6-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.

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F. Scheele, C. B. Lambalk, J. Schoemaker, H. van Kessel, J. de Koning, J. A. M. J. van Dieten, G. P. van Rees and T. J. M. de Vries Robles-Korsen

ABSTRACT

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