Division of Reproductive Biology, Department of Obstetrics and Gynaecology, University of Groningen, Hanzeplein 1, PO Box 30.001, 9700 RB Groningen, The Netherlands
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Abstract |
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Key words: antagonist/GnRH responsiveness/LH/oestradiol
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Introduction |
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These effects of FSH are attributed to the action of one single non-steroidal factor of ovarian origin: gonadotrophin-surge inhibiting or surge-attenuating factor (GnSIF or GnSAF), which suppresses the pituitary responsiveness to GnRH (de Koning, 1995; Fowler and Templeton, 1996
). GnSIF/AF is claimed to suppress the gonadotrophin surge by preventing GnRH sensitization of the pituitary gland for its own gonadotrophin-releasing action (Koppenaal et al., 1992
; Fowler et al., 1993
, 1994
; de Koning, 1995
; Byrne et al., 1996
): the so-called GnRH-self priming effect (Aiyer et al.; 1974). However, GnRH self-priming is not the only factor contributing to the increase in pituitary responsiveness to GnRH at the time of the pre-ovulatory GnRH surge; also oestradiol, secreted by the developing follicles, plays a role in this respect (Schuiling et al., 1987
, 1990
). Attenuation by FSH treatment of the pre-ovulatory gonadotrophin surge, therefore, may also be due to FSH-induced suppression of the augmenting effect of oestradiol. This suggestion was tested in cycling, pseudopregnant (PSP) and ovariectomized pseudopregnant rats (OVXPSP).
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Materials and methods |
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Pretreatment
A number of the rats received two s.c. Silastic implants containing oestradiol (Organon, Oss, The Netherlands) or two sham (empty) implants at times apparent from the `experimental protocol'. The dimensions of the implants were: length 1.0 cm; inside diameter 1.57 mm; outside diameter 3.18 mm. Such implants are known to significantly elevate plasma oestradiol concentrations in PSP rats (Schuiling et al., 1996).
FSH (Metrodin®; Serono Benelux, Den Haag, The Netherlands), 10 IU/injection, dissolved in saline, was administered i.p. at times apparent from the `experimental protocol'. FSH injections were given at 09.00. A dose of 10 IU/injection was chosen, based on the results of previous studies (de Koning et al., 1994). On the day of induction of gonadotrophin responses/blood sampling (see `experimental protocol'), rats received a cannula in the right carotid artery (09.00).
Induction of LH response
Two injections of GnRH (Cryptocur®; Hoechst AG, Frankfurt am Main, Germany) of 0.5 µg/100 g body weight each were administered through the cannula for induction of LH responses; the first one at 13.00 (t = 0); the second one at 14.00. At 1 h before administration of GnRH, rats were anaesthetized with phenobarbitone (Interpharm BV, Meppal, The Netherlands; 80 mg/kg body weight i.p.) in order to block (in pro-oestrous rats) the spontaneous LH surge. Blood samples for assay of LH were taken through the cannula at times apparent from Figure 1. Progesterone in PSP rats and oestradiol in all groups of rats were measured in the t = 0 sample. The plasma samples were stored frozen at 20°C until assay.
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Parameters
See Figure 1; LH responses were judged on the basis of the mean maximal increments of the plasma LH concentrations, which did not yield different results when compared with quantification using the area-under-the-curve as the parameter. This parameter was considered to reflect the pituitary responsiveness to GnRH (Schuiling et al., 1988
). Increments were calculated by subtracting the basal LH concentration at t = 0 from the maximal LH concentration (i.e. the primed LH response) induced by the second GnRH injection, at t = 80 min. The magnitude of the GnRH-self priming effect (`priming factor') was calculated by dividing the mean maximal increment of the LH response induced by the second GnRH injection by the mean maximal increment of the LH response induced by the first GnRH injection. The magnitude of the augmenting effect of oestradiol (`oestradiol-augmenting factor') was assessed by dividing the mean maximal increment of the LH response induced by the second GnRH injection of oestradiol-treated rats, by that of the corresponding control rats.
Experimental protocols
Experiment 1; cycling rats Spontaneous LH surges; effect of FSH:
Cycling rats received FSH or solvent injections on oestrus, di-oestrus-1 and di-oestrus-2. On pro-oestrus, blood samples for measurement of the spontaneous LH surges were taken at times apparent from Figure 2.
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Experiment 2: pseudopregnant rats Pituitary responsiveness to GnRH; effect of oestradiol and FSH:
Pseudopregnant rats received two oestradiol-implants or two sham implants on day 4 of PSP. Rats were treated with FSH or solvent on days 5, 6 and 7 of PSP. LH responses were induced on day 8; blood samples were taken at times apparent from Figure 1.
Experiment 3: ovariectomized PSP rats Pituitary responsiveness to GnRH; effect of oestradiol and FSH:
Rats were ovariectomized on day 4 of PSP (OVXPSP rats). Immediately after ovariectomy, rats received two oestradiol-implants or two sham implants. Rats were treated with FSH or solvent on days 5, 6 and 7 of PSP; LH responses were induced on day 8. Blood samples were taken at times apparent from Figure 1.
Statistical analysis
Data are expressed as means ± SEM. Statistical comparisons were made by analysis of variance (ANOVA), followed by Tukey's HSD test (Tukey, 1951). Pairs of groups were compared using the MannWhitney U-test. The level of significance was chosen at P < 0.05.
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Results |
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Pituitary responsiveness to GnRH; effect of oestradiol and FSH
Figure 3 shows the effect of FSH treatment on the LH responses, induced by two consecutive GnRH injections in pro-oestrous, phenobarbitone-blocked 4-day cycling rats. Rats were (right panel) or were not (left panel) pretreated with oestradiol; Figure 4
shows that oestradiol treatment significantly elevated the plasma oestradiol concentrations. Figure 3
shows that both before and after oestradiol-treatment, there was a strong GnRH-self priming effect (priming factor (for definition, see Materials and methods and Figure 1
): 5.1 ± 0.6 and 8.0 ± 3.1 respectively). There was no additional augmenting effect of oestradiol treatment on the pituitary responsiveness to GnRH (oestradiol-augmenting factor (see Figure 1,
0.8). FSH strongly suppressed the GnRH-induced LH response of both control rats and oestradiol-treated rats (P < 0.05; analysis of variance followed by Tukey's HSD test) by suppressing the augmentative effect of oestradiol (oestradiol-augmenting factor: 0.16). FSH, however, had no effect on the plasma oestradiol concentrations (Figure 4
) and did not suppress the GnRH-self priming effect (priming factors of FSH-treated animals before and after oestradiol treatment: 6.9 ± 1.1 and 9.1 ± 3.1 respectively).
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Discussion |
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Oestradiol strongly augmented the pituitary GnRHresponsiveness in PSP and OVXPSP rats, but not in cycling rats. The lack of an effect of exogenous oestradiol in cycling rats is probably due to the fact that in these animals the pituitary responsiveness is already augmented by endogenous oestradiol (Schuiling et al., 1987; 1990). FSH suppressed the pituitary GnRH-responsiveness of both oestradiol-treated and control PSP-8 rats, but not that of similarly treated OVXPSP rats, again suggesting that some ovarian factor is involved in the effect of FSH. Analysis of the data revealed that the suppressive effect of FSH treatment in oestradiol-treated cycling and PSP rats was caused by blockade of the augmenting effect of oestradiol; not by blockade of the GnRH-self priming effect or by an effect of oestradiol on the GnRH-self priming effect. This suggests that FSH stimulates the production of some factor which antagonizes the effect of oestradiol.
Suppression by an FSH-induced ovarian factor of the augmentative effect of oestradiol may also explain the suppressive effect of FSH on the pituitary GnRH-responsiveness of non-oestradiol-treated pro-oestrous rats, given the oestradiol-augmented pituitary GnRH-responsiveness of such animals. Similarly, it may explain the suppressive effect of FSH in control PSP rats, as such animals, too, have significant plasma concentrations of oestradiol, although these concentrations are lower than those of pro-oestrous rats. Even the fact that in OVXPSP rats the augmentative effect of oestradiol is stronger than in intact PSP rats can be explained by the suggestion that the ovaries produce a factor suppressing the effect of oestradiol.
The question arises whether this putative factor is identical to GnSIF/AF. GnSIF/AF has been claimed to antagonize both the GnRH-self priming effect (see Introduction) and to delay the timing of the maximal increase of the blood levels of LH (Koppenaal et al., 1993; de Koning et al., 1994
). As far as we know, however, antagonism by GnSIF/AF of the augmentative effect of oestradiol has not been reported, although Hodgen and co-workers (Sopelak and Hodgen, 1984
; Schenken and Hodgen, 1986
) and Templeton and co-workers (Messinis and Templeton, 1989
, 1990a
,Messinis and Templeton, b
; Messinis et al., 1994b
), observed in monkeys and women respectively, that GnSIF/AF suppressed the GnRH-responsiveness of oestradiol-treated individuals. In contrast to GnSIF/AF, the present factor did not induce delay of the pre-ovulatory LH-surge and did not antagonize the GnRH-self priming effect; it only antagonized the augmentative effect of oestradiol. GnSIF/AF and the `oestrogen-antagonizing factor' described in this paper, therefore, differ from each other in important aspects.
The present experiments show that under the influence of FSH, the ovaries not only produce oestradiol, but also a factor which antagonizes the effect of oestradiol on the pituitary gland. This putative `oestrogen-antagonizing factor' is not the only factor inhibiting the effect of oestradiol on the pituitary GnRH-responsiveness. In a previous study, it was demonstrated that the effect of oestradiol is also regulated by the plasma concentration of GnRH: the GnRH concentration dose-dependently suppresses the augmentative effect of oestradiol on the pituitary GnRH-responsiveness (Schuiling et al., 1996). On the other hand, by exerting a negative feedback effect on the hypothalamic GnRH secretion oestradiol itself controls the amount of GnRH exposure of the pituitary gland. It thus appears that a complex relationship exists between the effects of GnRH on the one hand and that of oestradiol on the other.
The present data suggest that the control of the effect of oestradiol on the pituitary gland is even more complex. Apparently, the effect of oestradiol is not controlled by one, but by (at least) two factors suppressing the pituitary responsiveness to GnRH: GnRH and the present putative `oestrogen-antagonizing factor'. This tight control of the pituitary GnRH-responsiveness with respect to the augmentative effect of oestradiol may be of particular importance in humans. During each ovulatory cycle, in order to avoid multiple pregnancies, the human ovaries should together preferably not produce more than one ovum. This probably demands gonadotrophin secretion rates which do not exceed certain limits. In the fine-tuning of the gonadotrophin secretion, the `oestrogen-antagonizing factor`, postulated in this paper, may play a role.
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Acknowledgments |
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Notes |
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References |
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Submitted on May 6, 1998; accepted on October 1, 1998.