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  • Author: A. M. I. Tijssen x
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D. W. Koppenaal, A. M. I. Tijssen and J. de Koning


The present study was designed to explore further the functional antagonism between gonadotrophin-releasing hormone (GnRH) and the ovarian factor, gonadotrophin surge-inhibiting factor (GnSIF). In all experiments, pituitary tissue was exposed to various amounts of GnSIF, after which the self-priming action of GnRH was studied. GnSIF was increased in vivo by FSH treatment and increased in vitro by adding various amounts of follicular fluid (FF) to cultured pituitary cells.

Treatment with 3 or 10 IU FSH suppressed the initial LH response and delayed the maximally primed LH response to GnRH. Treatment with FSH was only effective in intact rats on days 1 and 2 of dioestrus. There was no difference in the rate of maximal LH release irrespective of treatment with either FSH or saline. Since FSH treatment was ineffective in long-term ovariectomized rats, it was concluded that the initial suppressive effect of FSH on LH release was mediated by GnSIF.

Cycloheximide prevented the self-priming action of GnRH by inhibiting GnRH-induced protein synthesis. The initial protein synthesis-independent GnRH-stimulated LH release, which was already suppressed by FSH treatment, remained suppressed in the presence of cycloheximide. Pretreatment with GnRH in vivo increased the protein synthesis-independent GnRH-induced LH release during subsequent incubation of the glands. This increase did not occur after FSH treatment.

Pituitary cells, cultured for 20 h in medium only, failed to elicit the self-priming effect of GnRH. Preincubation with FF maintained the self-priming effect. This was independent of the concomitant presence of various amounts of oestradiol. Preincubation with bovine FF suppressed the initial GnRH-stimulated LH release dose-dependently. Porcine FF, human FF and testicular extract suppressed the release of LH in a similar way.

It was concluded that GnSIF suppresses the initial LH response to continuous GnRH stimulation. Increased levels of GnSIF caused by FSH treatment also delayed the primed LH release. The mechanism of functional antagonism between GnSIF and GnRH could give rise to the occurrence of the phenomenon of GnRH self-priming.

Journal of Endocrinology (1992) 134, 427–436

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D. W. Koppenaal, A. M. I. Tijssen, J. A. M. J. van Dieten and J. de Koning


Female rats were treated with Metrodin (highly purified urinary FSH from menopausal women) or saline during the oestrous cycle. On the day of pro-oestrus they were anaesthesized with phenobarbital and received four repetitive LHRH injections 1 h apart. This treatment with FSH suppressed the unprimed LH response to the first LHRH injection. During the subsequent injections the maximal LHRH self-priming was delayed by 3 h till the fourth LHRH stimulation. At this time, LH release in response to LHRH was equally as high as shown in the saline controls after the second LHRH injection. Ovariectomized rats did not show the self-priming effect and FSH treatment was ineffective in suppressing LHRH-induced LH release. Administration of FSH followed by an additional 4- or 24-h period before LHRH stimulation were equally effective in suppressing the unprimed LH release and delaying (up to 3 h) the maximal priming of LH release by LHRH. Even 4–20-fold increased amounts of LHRH did not affect the suppressed unprimed release of LH after FSH treatment. Treatment with FSH did not change oestradiol and progesterone levels.

It was concluded that FSH treatment suppresses the unprimed LHRH-induced LH release and delays maximal LHRH self-priming by enhancing the release of an ovarian factor.

Journal of Endocrinology (1991) 129, 205–211

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D. W. Koppenaal, J. A. M. J. van Dieten, A. M. I. Tijssen and J. de Koning


This study was designed to explore the efficacy of gonadotrophin-releasing hormone (GnRH) to antagonize the effect of gonadotrophin surgeinhibiting factor (GnSIF) on the timing of the induction by GnRH of the maximal self-priming effect on pituitary LH responsiveness. The GnSIF levels were increased by FSH treatment and reduced after gonadectomy.

Female rats were injected s.c. with 10 IU FSH or saline (control) on three occasions during the 4-day cycle. Serial i.v. injections of GnRH (500 pmol/kg body weight) were administered to intact rats on the afternoon of pro-oestrus or 15–30 min after ovariectomy. Intact male rats were given 10 IU FSH and 500 or 2000 pmol GnRH/kg body weight on an equivalent time-schedule. Endogenous GnRH release was suppressed with phenobarbital.

In intact female control rats, the timing of the maximally primed LH response was delayed as the GnRH pulse-interval increased. FSH treatment of female rats induced a suppression of the initial unprimed LH response and delayed the maximally primed LH response, which showed further delay as the GnRH pulse-interval was increased.

When the pulsatile administration of GnRH was started 15–30 min after ovariectomy, the priming effect of GnRH did not change as the GnRH pulse-interval was increased in the saline-treated rats. However, FSH treatment caused a suppression of the unprimed LH response, a delay in the primed LH response and decreased the delay of the maximally primed LH response to GnRH when the GnRH pulse-interval was decreased.

Increasing the interval between ovariectomy and the first GnRH pulse to 4 h diminished the efficacy of the FSH treatment: GnRH-induced priming was delayed by only one pulse instead of the two pulses in control rats.

In intact males but not in orchidectomized rats, a self-priming effect was demonstrated during GnRH pulses which were 1 h apart. The effect of 2 nmol GnRH/kg body weight was the most pronounced. Compared with intact female rats, the timing of the maximally primed LH response was delayed by 1 h. FSH treatment did not affect the pituitary LH response to both dose levels of GnRH.

It is concluded that FSH treatment increased the release of GnSIF by the ovary, then induced a state of low responsiveness of the pituitary gland to GnRH and subsequently delayed GnRH-induced maximal self-priming. The efficacy of GnRH to prime the pituitary gland was higher when GnSIF levels were decreasing after removal of the ovaries. On the other hand, GnSIF was more effective when the GnRH pulse-interval was increasing. This allows GnSIF more time to restore the unprimed state of the pituitary gland after each GnRH pulse-induced self-priming effect. It remains a matter of debate whether a similar mechanism of action is present in the male rat or whether this mechanism is suppressed by endogenous hormones such as androgens.

Journal of Endocrinology (1993) 138, 191–201

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


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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W. J. de Greef, J. de Koning, A. M. I. Tijssen and B. Karels


Treatment of ovariectomized rats with 50 μg oestradiol benzoate, followed by 20 μg oestradiol benzoate 3 days later, induced surges of LH and FSH on the day following the second injection with oestradiol benzoate. During this surge of gonadotrophins, which was not blocked by the anaesthetic required to collect hypophysial stalk blood, increased hypophysial stalk plasma levels of immunoreactive LHRH were noted. Furthermore, the levels of LHRH in hypophysial portal blood were found to fluctuate. Measurement of LHRH in a pool of portal plasma revealed similar results when determined by radioimmunoassay and by a sensitive in-vitro bioassay.

To mimic the observed release of LHRH during the surge of gonadotrophins, LHRH was infused, either systemically or directly into a long portal vessel, into oestrogen-treated, ovariectomized rats which had their endogenous release of LHRH blocked by pentobarbitone. An infusion of LHRH into the jugular vein, resulting in peripheral levels of LHRH which were somewhat lower than those measured in hypophysial stalk plasma, caused a surge of FSH similar to that found in rats used for collection of hypophysial stalk blood. When compared with the values in the latter animals, however, the levels of LH became two to four times higher by this infusion of LHRH. When LHRH was infused directly into a long portal vessel to mimic the observed secretion rate of LHRH during the oestrogen-stimulated surge of gonadotrophins, then the surges of LH and FSH were lower than those observed in the rats used for collection of stalk blood.

J. Endocr. (1987) 112, 351–359

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


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, 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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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.