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Search for other papers by G. E. Webley in
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ABSTRACT
The antigonadotrophic action of a prostaglandin F2α analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P<0·01), hCG-stimulated cAMP (40% decrease; P < 0·05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P < 0·01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge.
Journal of Endocrinology (1991) 131, 319–325
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ABSTRACT
The interaction between luteotrophic and luteolytic agents in controlling progesterone production by the marmoset corpus luteum in the late luteal phase/early pregnancy was investigated at the local level in vivo using a perfusion cannula system. Perfusion of the prostaglandin F2α(PGF2α) analogue, cloprostenol (0·5 μg/ml), resulted in an immediate fall in progesterone production. This response was not sustained in two out of five corpora lutea but pregnancy was terminated in all animals exposed to PGF2α. Perfusion of human chorionic gonadotrophin (hCG) (4 μg/ml) alone significantly stimulated progesterone secretion but there was no response to hCG when the corpus luteum had previously been perfused with PGF2α. Perfusion with hCG together with PGF2α prevented a fall in progesterone secretion. The results suggest that the luteolytic action of PGF2α in the marmoset may be to prevent luteotrophic support of the corpus luteum. Melatonin (860 pmol/l), perfused either with PGF2α or after PGF2α, stimulated progesterone production. The ability of melatonin to influence progesterone production by the primate corpus luteum may therefore be by both a direct luteotrophic action and the prevention of luteolysis. Application of the perfusion system in order to investigate the ability of deglycosylated hCG to antagonize the action of hCG at the corpus luteum showed the necessity of testing pure preparations of hormones.
J. Endocr. (1987) 114, 231–239
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ABSTRACT
The effect of human chorionic gonadotrophin (hCG) and melatonin on the local production of progesterone by the marmoset corpus luteum was investigated in vivo using a perfusion cannula system. Progesterone secretion was measured in 10-min fractions of buffer which had been perfused through the corpus luteum at a flow rate of 70 μl/min for a maximum of 3 h in anaesthetized animals. Two corpora lutea were cannulated in each animal; one for perfusion of test material and the other for perfusion with buffer alone as a control. Perfusion with hCG (25 i.u./ml), investigated as a positive control, produced a marked stimulation of progesterone secretion which increased 10–20 min from the start of perfusion and reached a peak after 30–60 min. A stimulation of progesterone was also observed after perfusion with melatonin (860 pmol/l). The response was evident within 10–30 min of the hormone reaching the corpus luteum and was similar in magnitude to that observed for hCG. The ability of melatonin to stimulate progesterone secretion supports previous in-vitro studies and suggests an ovarian action for melatonin in the primate. The local perfusion system described may have potential uses in studies of luteal function related to aspects of infertility or regulation of fertility.
J. Endocr. (1987) 112, 449–457
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In the grey squirrel pregnancy continues in the absence of the ovaries from day 16 of gestation. Since the investigation was performed on squirrels caught in the wild the stage of gestation was estimated from plasma progesterone levels, volume of corpora lutea at ovariectomy and date of parturition for those squirrels which did give birth. Three squirrels ovariectomized in early pregnancy did not give birth. Plasma progesterone levels after ovariectomy were lower than those found in normal pregnant squirrels suggesting that the corpora lutea are a continuing source of progesterone even at a time when pregnancy can continue in their absence.
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ABSTRACT
Progesterone production by dispersed luteal cells obtained from the marmoset monkey on day 14 after ovulation can be stimulated by both prostaglandin F2α (PGF2α) and its structural analogue, cloprostenol. To establish whether these responses can be attributed to cross-reaction with the prostaglandin E2 (PGE2) receptor, this study compared the involvement of cyclic adenosine-3′,5′-monophosphate (cAMP) and protein kinase C (PKC) in the luteotrophic responses to PGE2, PGF2α and cloprostenol. While all three prostaglandins stimulated similar increases in progesterone production (239·5 ± 7·9% of control; P <0·01), only PGE2 stimulated a significant increase in cAMP accumulation (373·2 ± 28·4% of control; P <0·01). This study is the first to demonstrate PKC activity in the marmoset ovary. Following down-regulation of PKC with a tumour-promoting phorbol ester, 4β-phorbol 12-myristate 13-acetate (4β-PMA), basal progesterone production was significantly increased (150·9 ± 8·2% of control; P <0·05) and the luteotrophic effects of PGF2α and cloprostenol were no longer evident, whereas the response to PGE2 was unaffected. These observations are consistent with the differential involvement of cAMP and PKC in the luteotrophic responses to PGE2 and PGF2α/cloprostenol respectively. Hence, we conclude that the luteotrophic actions of prostaglandins E2 and F2α on dispersed marmoset luteal cells are mediated via different receptors and signal transduction pathways.
Journal of Endocrinology (1993) 138, 291–298
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Search for other papers by K. P. Willey in
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ABSTRACT
The role of melatonin in animals which do not show marked seasonal changes in reproduction is disputed, in part because of the wide variation in reported concentrations. One reason for this may be the difficulties associated with the measurement of low molar concentrations of melatonin and the presence of a wide variety of potentially cross-reacting substances. The availability of a high affinity antiserum has allowed an assay, with low cross-reactivity and good sensitivity, to be established for the direct measurement of melatonin in a wide range of biological fluids, in particular serum, plasma and follicular fluid from man and rat. The high affinity of the antiserum enabled a tritium label of high specific activity to be used, removing the problems associated with the iodination of a small molecular weight compound. Melatonin concentrations in the assay were evaluated by four different methods: UV absorbance, gas chromatography, comparison of the immunoreactive concentrations of the label with the expected concentration by dilution and by comparison with a previously established assay which uses the same antiserum.
Melatonin was measured in serum from twelve healthy women over two 24-h periods; eight women with normal menstrual cycles and four taking the contraceptive pill. Concentrations were found to range from 19·8 to 215 pmol/l during the day in both groups. In women with normal menstrual cycles peak concentrations of 513·2 ± 54·1 (s.e.m.) pmol/l were recorded at 04.00 h, whereas higher concentrations were found in women taking the pill, reaching a peak of 849·12 ± 21·8 (s.e.m.) pmol/l at 04.00 h. Similar melatonin concentrations were measured in the two 24-h periods.
In the adult male rat, serum melatonin concentrations varied from 92·66 ± 37·9 (s.e.m.) pmol/l at 12.00 h, rising to 526 ± 55·6 (s.e.m.) pmol/l at 04.00 h.
This direct assay is more practical and robust than the assays currently available. The careful validation of assay characteristics allows its widespread use in both clinical studies and the investigation of the role of melatonin in different species.
J. Endocr. (1985) 106, 387–394
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ABSTRACT
Peripheral concentrations of immunoreactive (ir) inhibin have been measured during the ovarian cycle and early pregnancy in the marmoset monkey. Blood samples were taken (three per week) during conception (n = 6) and non-conception (n = 5) cycles. Ir-inhibin was measured by radioimmunoassay using an antiserum raised against a synthetic peptide fragment of the α subunit of human inhibin. Monomeric bovine α subunit and 32 kDa bovine inhibin were used as tracer and standard respectively. In all animals low concentrations of ir-inhibin were recorded during the follicular phase (40–60 μg/l) of the cycle. After ovulation, ir-inhibin concentrations increased but the peak concentrations attained differed between conception and non-conception cycles. In non-pregnant animals ir-inhibin concentrations reached a maximum of 242±16 μg/l on days 12/13 after ovulation. In pregnant animals ir-inhibin concentrations were significantly (P < 0·05) higher (1·8-fold) than in non-pregnant animals on days 8/9 after ovulation, and reached a maximum value of 636±141 μg/l on days 20/21 after ovulation. Administration of an LHRH antagonist during the luteal phase on days 6–8 after ovulation resulted in a significant (P < 0·05) decrease in progesterone and ir-inhibin concentrations within 4 and 8 h respectively. This was prevented by co-administration with human chorionic gonadotrophin. Administration of cloprostenol to pregnant animals between days 17 and 20 after ovulation halved the initial concentrations of both inhibin and progesterone within 1·5 h. The increase in plasma ir-inhibin concentrations in the luteal phase and the apparent similarity in control of ir-inhibin and progesterone supports a luteal source of ir-inhibin in both conception and non-conception cycles. The higher levels of ir-inhibin from days 8/9 after ovulation in conception cycles were not related to any detectable increase in peripheral concentrations of chorionic gonadotrophin and occurred at least 4 days before the expected time of implantation. This suggests a role for the conceptus in inhibin secretion which may involve the release of an embryo message before implantation.
Journal of Endocrinology (1991) 128, 465–473
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ABSTRACT
The effects of the luteolytic and luteotrophic agents cloprostenol, human chorionic gonadotrophin (hCG) and melatonin on the corpus luteum have been investigated in marmoset monkeys treated with an LHRH antagonist to reduce endogenous LH secretion. This has allowed the effects of these agents to be investigated in the absence of the principal endogenous luteotrophin.
Administration of the LHRH antagonist ([N-acetyl-dβNal1-d-pCl-Phe2-d-Phe3-d-Arg6-Phe7-Arg8-d-Ala10]NH2-LHRH) or cloprostenol between days 7 and 11 after ovulation (preimplantation) resulted in luteolysis. A significant (P<0·05) decrease in progesterone concentrations had occurred by 4 h after administration of the LHRH antagonist and was indeed preceded by a fall in LH concentrations. Coadministration of hCG with the LHRH antagonist prevented the fall in progesterone. In contrast, administration of cloprostenol resulted in an immediate fall in progesterone concentrations, to less than half the initial level within 1 h, and co-administration with hCG did not prevent the fall. Administration of hCG stimulated progesterone production when given 8 h after the LHRH antagonist but not after 24 h. Cloprostenol prevented the stimulation by hCG. Co-administration of melatonin with the LHRH antagonist did not prevent the decrease in progesterone concentrations. Melatonin was also not effective in preventing the fall in progesterone induced by cloprostenol. However, co-administration of melatonin and cloprostenol between days 17 and 21 after ovulation (post-implantation) significantly (P<0·05) delayed the fall in progesterone seen with cloprostenol alone.
These results suggest that while the LHRH antagonist and cloprostenol have different sites of action their effect is similar at the corpus luteum, that is in depriving the corpus luteum of luteotrophic support. The results also suggest that melatonin may be able to influence the luteolytic action of cloprostenol but that its effect varies with the stage of the cycle. The physiological role for such an action, if any, remains unknown.
Journal of Endocrinology (1991) 128, 121–129