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ABSTRACT
In goldfish, dopamine acts as an endogenous inhibitor of basal and gonadotrophin-releasing hormone (GnRH)-stimulated gonadotrophin release. The purpose of the present study was to investigate the effects of dopamine on the pituitary GnRH receptors in vivo and in vitro in goldfish. The goldfish pituitary contains two classes of GnRH-binding sites, a high-affinity/low-capacity site and a low-affinity/high-capacity site. Injection of domperidone, a dopamine antagonist, resulted in a dose- and time-related increase in capacity of both the high- and low-affinity GnRH-binding sites; apomorphine, a dopamine agonist, completely reversed this effect. The effects on GnRH receptor capacity correlated very closely with changes in serum gonadotrophin concentrations. Domperidone was generally without effect on GnRH-binding affinity; however, a small but significant decrease in affinity was observed for the low- affinity binding site at 18 h after injection of the highest dose of domperidone used (40 μmol/kg body weight). Treatment with apomorphine of goldfish pituitary fragments in a perifusion system caused a decrease in the capacity of both the high- and low-affinity GnRH-binding sites without affecting binding affinity; domperidone reversed this effect. It is concluded that the dopaminergic inhibition of basal and GnRH-stimulated gonadotrophin release in goldfish might, in part, be the result of a down-regulation of the pituitary GnRH receptors; this effect of dopamine can be achieved by a direct action at the pituitary level.
Journal of Endocrinology (1989) 121, 239–247
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Abstract
Immunochemical studies were undertaken to identify surface-orientated epitopes of the free α subunit of human chorionic gonadotrophin (hCG-α) at the amino acid sequence level. We investigated the molecular organization of these epitopes, resolved the immunological topography in terms of spatial arrangement of antigenic domains and related structures to functions such as subunit association or receptor binding. Overlapping synthetic peptides covering the entire amino acid sequence of hCG-α, an enzymatically digested hCG-α subunit, and a reduced and alkylated hCG-α preparation were assayed in a solid-phase one-site enzyme-linked immunoassay, and in a solution-phase competitive radioimmunoassay (RIA). The antigenic topography was mapped by monoclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). On the surface of hCG-α, seven different epitopes (α1–α7), arranged in four spatially distinct domains, could be distinguished: A, α1,2,4; B, α3,5; C, α6; D, α7. The peptides spanning hCG-α(13–18), hCG-α(17–22) and hCG-α(33–42) appeared to contribute to the formation of epitopes α2, α4 and α6 respectively. Since epitope α6 is present only on the free non-assembled subunit of different species, we concluded that the region hCG-α(33–42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (α7) are destroyed by reducing and alkylating hCG-α. In contrast, chymotryptic digestion of hCG-α, leading to release of the heptapeptide hCG-α(41–47), did not affect epitope expression, indicating that this sequence is not involved in the formation of antigenic determinants. Addressing the biological properties of hCG-α epitopes by radioreceptor assay revealed that the three hCG-α peptides corresponding to epitopes α2, α4 and α6 did not displace radiolabelled hCG from its receptor, whereas any of the MCAs directed against determinants (α1–α5), shared by hCG and hCG-α, totally inhibited binding. Consistent with this, the antibodies neutralized the biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that hormone antibody-binding sites differ from those of hormone receptor binding, revealing no essential congruence of immunologically and biologically active domains.
Journal of Endocrinology (1994) 140, 145–154
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Abstract
The effects of a single injection of recombinant human FSH (rhFSH; Org32489) on ovulation rate and timing and on antral follicle growth were studied in adult 5-day cyclic rats. Rats injected at 1700 h on dioestrus-2 with a dose of 10 IU rhFSH showed, on average, no increase in ovulation rate on the day of expected oestrus. However, an additional, precocious ovulation resulting in a normal number of corpora lutea 13·3±0·4, n=6) was found to take place on the night after injection, i.e. dioestrus-3. No mating behaviour, as shown by the absence of vaginal plugs the next morning, was observed at this ovulation. Follicle counts showed a loss of large antral follicles due to ovulation and increased numbers of healthy small antral follicles at 17 and 41 h after injection, indicating a decrease of atresia of growing follicles as well as additional recruitment of new antral follicles. The endogenous serum FSH concentration on the subsequent day of oestrus (65 h after the rhFSH injection) as well as recruitment of small antral follicles were lower in the rhFSH-treated rats than in saline-treated controls. The ovulation at oestrus, 48 h after the precocious, rhFSH-induced ovulation showed large differences in the number of oocytes between the rats in one treatment group.
Similar results in terms of immediate ovulation induction were obtained by using a highly purified human urinary FSH preparation (i.e. metrodin). Furthermore, the direct induction of ovulation by rhFSH or metrodin could not be prevented by the injection of an LHRH antagonist.
It was concluded that rhFSH can induce acute ovulation in rats, and stimulates follicular development directly or indirectly through increased FSH levels after ovulation. It induces antral follicle growth and decreases early atresia in small antral follicles.
Journal of Endocrinology (1995) 144, 39–47
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Abstract
To investigate whether the progesterone antagonist RU486 has a direct effect on ovarian function, it was administered to immature female rats rendered hypogonadotrophic by administration of an LHRH antagonist and in which follicle development was stimulated by recombinant human FSH (recFSH).
In the first experiments the effects of LHRH antagonist and recFSH on follicle growth were evaluated. Female rats of 22 days of age were injected with an LHRH antagonist (Org 30276; 500 μg/100 g body weight) every other day. This treatment resulted in a tenfold decrease in serum LH concentrations and a twofold decrease in serum FSH concentrations at day 30 and caused a reduction in the number and size of antral follicles. Treatment with recFSH (Org 32489) twice daily from day 26 for 4 days in a total dose ranging from 5 to 20 IU/animal increased the number and size of antral follicles in a dose-related manner and resulted after 20 IU recFSH in a tenfold increase in the concentration of inhibin in serum and ovaries at day 30. Once it was established that LHRH antagonist treatment in immature rats could be used to study the effects of gonadotrophins or steroids on follicle function, this animal model was used to study the effects of RU486 on the ovary. RU486 was administered (twice daily for 4 days, 1 mg/injection) to LHRH antagonist-treated rats in which follicular growth and differentation were stimulated by 10 IU recFSH or by 10 IU recFSH plus 0·5 IU human chorionic gonadotrophin (hCG). RU486 had no effect on circulating levels of LH and FSH, but stimulated follicular atresia both in rats treated with recFSH alone and in rats treated with recFSH and hCG. Inhibin concentrations both in serum and ovaries were significantly increased after hCG treatment. RU486, however, did not increase inhibin in the rats treated with recFSH and in those treated with recFSH and hCG.
In summary, the present study has demonstrated that (1) immature rats treated with an LHRH antagonist can be used to study the effects of gonadotrophins and steroids on follicular function and (2) RU486 has a direct stimulatory effect on follicular atresia.
Journal of Endocrinology (1996) 150, 85–92