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G. A. Schuiling, H. Moes and T. R. Koiter

Abstract. The effect of a combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and oestradiol benzoate (EB) on the autonomous and the 'supra-maximally' LRH-stimulated in vitro release of LH and FSH by pituitary glands of 2 weeks ovariectomized (OVX) rats was studied using a perifusion system. The concentration of LRH in the perifusion medium was 1 μg/ml.

Pre-treatment with LRH during 6 days was effected by means of sc implanted Alzet® osmotic minipumps (MP). Control rats received a piece of silastic with the dimesions of a minipump ('sham-pump'; Sh-P). EB, 3 μg/injection or solvent (arachis oil) was sc injected on days –3 and –1 (day of perifusion: day 0).

Of the pituitary glands of EB-injected, Sh-P-implanted rats both the autonomous and the LRH-stimulated secretion of LH and the LRH-stimulated secretion of FSH were significantly higher than those of the oil-injected, Sh-P-implanted rats without EB administration. Pretreatment with LRH for 6 days had a suppressing effect on the autonomous and the LRH-induced depletion of the pituitary LH and FSH stores. In combination with EB, the suppressing effect of LRH pre-treatment on the LRH-stimulated secretion of LH and FSH was still greater: the pituitary gland appeared to be fixed in a relatively unresponsive state with very low autonomous LH and FSH secretion.

It is discussed that increase of pituitary LRH-responsiveness due to EB demands withdrawal of the pituitary gland from the influence of LRH, an effect which is in vivo achieved by the negative feedback of oestrogen on the hypothalamus.

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G. A. Schuiling, H. Moes and T. R. Koiter

Abstract. Three-weeks ovariectomized rats were sc implanted with Alzet® osmotic minipumps which released either LRH or the LRH-analogue buserelin at the rate of 250 ng/h. Control rats were implanted with a silastic 'shampump'. After explantation, 6 days later, the pituitary glands of part of these rats were exposed to the maximally active LRH concentration of 1 μg/ml for a period of 6 h. using a perifusion system. In a second group of rats explantation and perifusion was done not directly, but 5 days after cessation of the I.RH pretreatment.

After 6 days in vivo pre-treatment with LRH or with buserelin the pituitary LH and FSH stores were partially depleted, the depletion after buserelin being stronger than after LRH. The pituitary glands of the first group of rats showed rates of both maximally LRH-stimulated and unstimulated (autonomous) LH- and FSH-secretion which were strongly impaired, the impairment after buserelin being stronger than after LRH. In the group with a 5 days interval between in vivo LRH/buserelin pre-treatment and explantation the pituitary LH and FSH stores were restored to the range of pre-treatment levels. Of these pituitaries the autonomous secretion of LH and FSH as well as the maximally LRH-stimulated secretion of FSH was restored to the normal level; the maximally LRH-stimulated secretion of LH, however, remained depressed, indicating that 5 days after cessation of exposure to LRH or to buserelin, and in spite of restored pituitary LH/FSH contents, the sensitivity of the LH releasing system to LRH was still subnormal.

The results suggest that the autonomous secretion of LH and FSH as well as the LRH-stimulated secretion of FSH, but not the LRH-stimulated secretion of LH may be dependent on the content of the pituitary LH and FSH stores. Furthermore, after treatment with LRH or buserelin the autonomous secretion of LH may return to a normal level when the sensitivity of the LH releasing system to LRH is still impaired: apparently, the mechanisms underlying the autonomous and the LRH-stimulated LH secretion do not influence each other.

It is discussed that in situations in which a complete suppression of the pituitary-gonadal axis is demanded (carcinomata of the breast or the prostate; precocious puberty) desensitization of the pituitary gland with super-active LRH-analogues like buserelin alone is not sufficient, as this does not affect the autonomous secretion of LH and FSH. For total suppression of gonadal activity the pituitary gland must be completely depleted with relatively large doses of analogue.

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G. A. Schuiling, H. Moes and T. R. Koiter

Abstract. The effect of a combined in vivo pre-treatment with luteinizing hormone-releasing hormone (LRH) and either oestradiol benzoate (OB), clomiphene (-citrate) or OB plus clomiphene on the autonomous and the supramaximally LRH-stimulated in vitro secretion of LH and FSH by pituitary glands of long-term ovariectomized (OVX) rats was studied using a hemi-pituitary perifusion system. The concentration of LRH in the perifusion medium was 1 μg/ml. Pre-treatment with LRH during 5 days was effected by means of sc implanted Alzet® osmotic minipumps; control rats received a piece of silastic with the dimensions of a minipump. OB, 3 μg/injection, clomiphene 100 μg/injection or solvent were given on days 2 and 4 (day of perifusion: day 5). In rats not pre-treated with LRH neither OB, nor clomiphene changed the content of the pituitary gonadotropin stores. There was only a small but significant positive effect of the combined treatment with OB and clomiphene on the pituitary FSH content. LRH (partly) depleted the gonadotropin stores. This effect of LRH was potentiated by OB, but not by clomiphene. Clomiphene prevented the depletion-potentiating effect of OB. OB raised the LRH-stimulated secretion of LH and FSH as well as the autonomous secretion of LH. Clomiphene raised the LRH-stimulated (not the autonomous) secretion of LH and FSH. OB plus clomiphene had the same effect as OB alone. Clomiphene also raised the LRH-stimulated secretion of LH and FSH after pre-treatment with LRH, but OB did not do so: LRH prevented the stimulatory effect of OB but not of clomiphene. OB plus clomiphene had the same effect as OB alone. The absence of a stimulatory effect of OB on the LRH-stimulated secretion of LH and FSH in the LRH-pretreated rat appeared to be due to the very low gonadotropin content of the pituitary glands after pre-treatment with LRH and OB: the effect of OB on the LRH-responsiveness proper (i.e. release of LH and FSH as related to the pituitary LH and FSH content) remained stimulatory. Also clomiphene enhanced the LRH-responsiveness proper, but this drug cannot potentiate the gonadotropin stores-depleting effect of LRH. These results demonstrate that clomiphene exclusively 'behaves' like an oestrogen-agonist, able to enhance the LRH-stimulated gonadotropin secretion. Also in the LRH-pre-treated rat clomiphene acts like an oestrogen-agonist, but unlike oestradiol clomiphene cannot potentiate the LRH-induced depletion of the pituitary gonadotropin stores. Therefore, it can also raise the LRH-stimulated secretion of LH and FSH in the LRH-pre-treated OVX rat.

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G. A. Schuiling, H. Moes and T. R. Koiter

Abstract. The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment.

Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 μg EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRHstimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9.

On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores.

On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values.

In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH. After 5 days of in vivo pretreatment with LRH only, the in vitro non-stimulated and supra-maximally LRH-stimulated secretion of both LH and FSH were strongly impaired, the effect correlating well with the LRH-induced depletion of the pituitary LH/FSH stores. In such LRH-pretreated rats EB had on day 5 a negative effect on the (already depressed) LRH-stimulated secretion of LH (not on that of FSH). EB had no effect on the non-stimulated LH/FSH secretion. It could be demonstrated that the negative effect of the combined LRH/EB pretreatment was mainly due to the depressing effect of this treatment on the pituitary LH and FSH stores: the effect of oestradiol on the pituitary LRH-responsiveness (release as related to pituitary gonadotrophin content) remained positive. In LRH-pretreated rats, however, this positive effect of EB was smaller than in rats not pretreated with LRH.

Four days after removal of the minipumps there was again a positive effect of EB on the LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. The positive effect of EB on the pituitary LRH-responsiveness was as strong as in rats which had not been exposed to exogenous LRH. The non-stimulated secretion of FSH was again not affected by EB.

The results demonstrate that the effect of EB on the oestrogen-sensitive components of gonadotrophin secretion consists of two components: an effect on the pituitary LRH-responsiveness proper, and an effect on the pituitary LH/FSH stores. The magnitude of the effect of EB on the LRH-responsiveness is LRH dependent: it is very weak (almost zero) in LRH-pretreated rats, but strong in rats not exposed to LRH as well as in rats of which the LRH-pretreatment was stopped 4 days previously.

Similarly, the effect of EB on the pituitary LH and FSH stores is LRH-dependent: in the absence of LRH, EB has no influence on the contents of these stores, but EB can potentiate the depleting effect of LRH on the LH/FSH-stores. Also this effect disappear after cessation of the LRH-pretreatment.

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T. R. Koiter, H. Moes and G. A. Schuiling

Abstract. Six agonistic derivatives of GnRH, four of which have already been evaluated in clinical trials, were compared with GnRH itself in an in vitro test system (incubation of rat pituitary glands). It was investigated 1) how the release of LH was affected when the pituitary glands were incubated in the presence of these analogues or GnRH, and 2) how the release of LH continued after removal of the analogues or GnRH from the medium. It was also investigated how an in vivo pretreatment for 6 days with several doses of 5 of these analogues or GnRH affects 3) the plasma concentration of LH, 4) the pituitary content of LH, and, in vitro, 5) the autonomous and 6) agonist-stimulated secretion of LH. Each of the analogues showed for each of the six investigated parameters a 10- to 100-fold higher potency than GnRH itself. Between the six analogues there were only minor differences. It is discussed how the six investigated parameters may be the expression of one single property of all these analogues, namely a long retention in the pituitary gland with a strong binding to the GnRH receptor.

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G. A. Schuiling, H. Moes, J. de Koning and T. R. Koiter

Abstract. Ovariectomized rats were infused with varying doses of luteinizing hormone-releasing-hormone (LRH). Some of the rats were also treated with oestradiol benzoate (EB). The effects of these pre-treatments on the in vitro release of luteinizing hormone (LH) were studied. The following parameters of in vitro LH release were measured: a) the autonomous secretion rate; b) the secretion rate following maximum stimulation with LRH, and c) the total quantity of LH released during the 6-hour experiment.

The in vivo pre-treatments with LRH and EB dose-dependently decreased the pituitary LH content as well as all three of the above parameters of in vitro LH secretion. There was a linear relationship between the pituitary LH content and the three parameters of in vitro LH release. These parameters were therefore expressed as percentage of the pituitary LH content to give the relative LH secretion rates. The three parameters were thereby corrected for LRH/EB-induced changes in the pituitary LH content. In this way we obtained information on the effects of LRH and EB on the state of the LH release mechanisms of the gonadotropes.

EB potentiated the LRH-induced depletion of the pituitary LH stores at all in vivo LRH infusion rates. The effect of EB on the quantity of LH released during perifusion in vitro, however, varied with the previous LRH infusion rates. After LRH infusion rates lower than about 120 ng/h (which establishes plasma concentrations of about 70 ng/l) EB enhanced the stimulated quantity of LH released. After higher rates of LRH infusion, EB lowered the amount of LH released. The effect of EB on the relative secretion of LH in vitro, i.e. on the LH release mechanisms, however, was positive irrespective of the prior in vivo LRH infusion rates, although the effect of EB was greater at the lower rates of LRH infusion.

The effect of EB on the autonomous, in vitro, LH secretion rate was positive irrespective of the prior in vivo LRH infusion rates. The positive effect of EB on the mechanism underlying this component of LH secretion was LRH-independent.

The effect of EB on the mechanism underlying the LRH-stimulated component of LH release appeared to be strongly LRH-dependent. The effect of EB was maximal if the LRH infusion rate had been lower than about 50 ng/h. With higher infusion rates it became increasingly smaller and was zero at the rate of about 180 ng/h or more. The LRH infusion rates of 50 and 180 ng/h establish plasma LRH concentrations of about 30 and 90 ng/l. Thus, the positive effect of EB on the LRH-stimulated component of LH secretion can be regulated by LRH at the plasma concentration interval of 30–90 ng/l.

This study demonstrates that the 'overall' effect of EB on the LH secretion rate is determined by the 'balance' between the effect of EB on the pituitary LH content (the potentiation of the LRH-induced depletion of the LH stores) and the effect of EB on the LH release mechanisms (which effect, in the case of the LRH-stimulated component of LH secretion, can be suppressed by LRH). If the former effect dominates, the effect of EB on the secretion of LH is negative, if the latter dominates, the effect of EB is positive. The LRH concentration at which the positive effect turns into the negative effect is about 70 ng/l.

We suggest that the ability of LRH and EB to influence each others' effect on the pituitary gland at physiological concentrations of the two hormones, plays a role in the regulation of the secretion of LH.

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AG Nieuwenhuizen, GA Schuiling, SM Liem, H Moes, TR Koiter and JT Uilenbroek

Treatment of cyclic and pregnant rats with progesterone stimulates cell proliferation within the islets of Langerhans. It was investigated whether this effect of progesterone depends on sex and/or the presence of the gonads or the presence of oestradiol. For this purpose, Silastic tubes containing progesterone were inserted s.c. in intact and gonadectomized male and female rats, and in gonadectomized female rats treated with oestradiol. After 6 days of progesterone treatment, rats were infused for 24 h with 5-bromo-2'-deoxyuridine (BrdU) and dividing cells were identified in pancreatic sections by immunostaining for BrdU. Progesterone treatment increased islet-cell proliferation in intact male and female rats (P < 0.05), but not in gonadectomized male and female rats or in gonadectomized female rats supplemented with oestradiol. Furthermore, in intact male and female rats, progesterone treatment also stimulated cell proliferation in extra-islet pancreatic tissue (P < 0.05). Identification of the proliferating cells, by double-immunocytochemistry, revealed that progesterone treatment stimulated proliferation of both alpha and beta cells within the pancreatic islets. In extra-islet pancreatic tissue, progesterone treatment stimulated proliferation in both duct (cytokeratin 20-immunoreactive) and non-duct cells. Progesterone treatment did not increase the number of single glucagon or insulin-containing cells outside the pancreatic islets, nor that of cytokeratin 20/insulin double-positive cells, suggesting that progesterone treatment did not stimulate differentiation of duct cells into endocrine cells. Progesterone treatment did not affect insulin responses to an i.v. glucose load (0.5 g/kg body weight). It is concluded that progesterone stimulates pancreatic cell proliferation indirectly; gonadal factor(s), not identical to oestradiol, is (are) probably involved.