The relationship between the production and the anti-gonadotrophic action of prostaglandin F 2 alpha in luteal cells from the marmoset monkey (Callithrix jacchus) in the early and mid-luteal phase.

To address the potential luteolytic role for prostaglandin F(2 alpha) (PGF(2 alpha)) in the corpus luteum of the common marmoset monkey (Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF(2 alpha) was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF(2 alpha) synthesis. We also assessed the effects of the PGF(2 alpha) analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48-72 h (P<0.001). Intra-luteal PGF(2 alpha) concentrations were 5-fold higher in luteal cells isolated in the early luteal phase than in mid-luteal phase cells (16.5+/-3.5 versus 3.5+/-0.6 pmol/10(5) cells). While PGF(2 alpha) production was unaffected by hCG in vitro, it was decreased by indomethacin (1000 ng/ml) (P<0.05) and stimulated by the calcium ionophore A23187 (10 micromol/l) (P<0.05) in luteal cells from both stages of the luteal phase. Phospholipase A(2) did not influence PGF(2 alpha) production by day 3 luteal cells whereas at 10 IU/ml, it significantly stimulated PGF(2 alpha) production by day 14 luteal cells (P<0.05). Hence, the timing of luteolysis in the common marmoset monkey appears to involve changes in both the luteal cell response to and production of PGF(2 alpha).

The circadian pattern of melatonin and its positive relationship with progesterone in women.

To investigate the possible role of melatonin in the regulation of the human menstrual cycle, the circadian pattern of melatonin was determined in the follicular and luteal phases of 10 normal women. Four-hourly sampling was used to derive a melatonin index which described the total exposure to melatonin for 24 h. This sampling procedure adequately represented the circadian melatonin output and demonstrated that pulses of melatonin secretion, inconsistent with a measured half-life of 47 min, did not exist. A significant increase (P less than 0.001) in the melatonin index was found in the luteal phase compared to that in the follicular phase. To investigate the influence of exogenous progestins on the melatonin pattern, repeated 24-h profiles were measured in 8 women taking the 3-phase contraceptive pill. There was a significant increase (P less than 0.01) in the melatonin index associated with an increase in the dose of progestin. These results are consistent with a positive relationship between melatonin and progesterone and suggest that changes in the circadian pattern of melatonin secretion, rising during the luteal phase with a fall before ovulation, may act as a modulator of cyclicity.

PIP: 10 normal women participated in a study designed to investigate the possible role of melatonin in the regulation of the human menstrual cycle. The circadian pattern of melatonin was determined in the follicular and luteal phases of the study subjects who had conventional life styles and normal sleep/wake rhythms. Also investigated was the influence of exogenous steroids on the circadian pattern of melatonin, using subjects who were taking a 3-phase oral contraceptive (OC). Initially, 2 studies were performed to determine the frequency of sampling necessary to define the circadian profile of melatonin and to determine the relevance of that sampling frequency to the half-life of melatonin. 3 routes of administration were employed: 2 mg melatonin as a 0.04% solution in corn oil containing 2% ethanol, taken orally as a suspension in 50 ml milk; 2 mg melatonin in an 8% solution of ethanol in a nasal spray; and 2 mg melatonin in 250 mg ascorbic acid, taken orally in a gelatin capsule. A program of 4-hourly sampling was adopted. The 10 healthy women ranged in age from 20-40 years and had normal menstrual cycles. A circadian pattern of serum melatonin concentrations was observed in all study subjects. Figure 1 shows the 24-hour profiles of melatonin in 1 woman studied in the follicular and luteal phases of her menstrual cycle. The concentrations of melatonin in samples taken at intervals of 15 minutes for a 24-hour period indicated that episodic release did not take place. The increase in the melatonin index from the follicular to the luteal phase was highly significant for the group as a whole. The investigation of other aspects of the circadian pattern of melatonin showed a significant increase in the luteal phase compared to the follicular phase in peak serum melatonin concentrations, estimated from data at 4-hourly intervals. The mean duration of elevation was 11.6 +- 0.8 h in the luteal phase and 10.7 +- 0.7 h in the follicular phase. All subjects had a single peak concentration, at either 2400 or 0400 h. A change in the melatonin index was found in the 8 women using the 3-phase OC pill when sampled at 2 times during the cycle. A significant increase in the melatonin index was associated with an increase in the dose of progestin, either from no progestin intake to a low-dose or from a low-dose to a higher dose.

Local production of progesterone by the corpus luteum of the marmoset monkey in response to perfusion with chorionic gonadotrophin and melatonin in vivo.

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 microliters/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.

Regulation of the corpus luteum of early pregnancy in the marmoset monkey: local interactions of luteotrophic and luteolytic hormones in vivo and their effects on the secretion of progesterone.

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 F2 alpha (PGF2 alpha) analogue, cloprostenol (0.5 microgram/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 PGF2 alpha. Perfusion of human chorionic gonadotrophin (hCG) (4 micrograms/ml) alone significantly stimulated progesterone secretion but there was no response to hCG when the corpus luteum had previously been perfused with PGF2 alpha. Perfusion with hCG together with PGF2 alpha prevented a fall in progesterone secretion. The results suggest that the luteolytic action of PGF2 alpha in the marmoset may be to prevent luteotrophic support of the corpus luteum. Melatonin (860 pmol/l), perfused either with PGF2 alpha or after PGF2 alpha, 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.

Effect of ovariectomy on the course of gestation in the grey squirrel (Sciurus carolinensis).

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 pregnancy squirrels suggesting that the corpora lutea are a continuing source of progesterone even at a time when pregnancy can continue in their absence.

Prior exposure to gonadotrophins prevents the subsequent antigonadotrophic actions of cloprostenol by a cyclic AMP-dependent mechanism in cultured human granulosa cells.

The antigonadotrophic action of a prostaglandin F2 alpha 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 less than 0.01), hCG-stimulated cAMP (40% decrease; P less than 0.05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P less than 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.

Validation of a sensitive direct assay for melatonin for investigation of circadian rhythms in different species.

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 measurements 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.(ABSTRACT TRUNCATED AT 250 WORDS)

The luteotrophic actions of prostaglandins E2 and F2 alpha on dispersed marmoset luteal cells are differentially mediated via cyclic AMP and protein kinase C.

Progesterone production by dispersed luteal cells obtained from the marmoset monkey on day 14 after ovulation can be stimulated by both prostaglandin F2 alpha (PGF2 alpha) 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, PGF2 alpha 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, 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA), basal progesterone production was significantly increased (150.9 +/- 8.2% of control; P < 0.05) and the luteotrophic effects of PGF2 alpha 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 PGF2 alpha/cloprostenol respectively. Hence, we conclude that the luteotrophic actions of prostaglandins E2 and F2 alpha on dispersed marmoset luteal cells are mediated via different receptors and signal transduction pathways.

Increased concentrations of immunoreactive inhibin during conception cycles in the marmoset monkey: suppression with an LHRH antagonist and cloprostenol.

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 alpha subunit of human inhibin. Monomeric bovine alpha 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 micrograms/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 micrograms/l on days 12/13 after ovulation. In pregnant animals ir-inhibin concentrations were significantly (P less than 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 micrograms/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 less than 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the luteolytic action of gonadotrophin-releasing hormone antagonist and cloprostenol, and the ability of human chorionic gonadotrophin and melatonin to override their luteolytic effects in the marmoset monkey.

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-D beta Nal1-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 less than 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 less than 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.

Prostaglandin F2 alpha stimulates cAMP phosphodiesterase via protein kinase C in cultured human granulosa cells.

To investigate the involvement of cyclic AMP phosphodiesterase in the antigonadotrophic actions of prostaglandin F2 alpha (PGF2 alpha), human granulosa cells were cultured in serum-supplemented medium for 72 h, treated for 30 min with cloprostenol or phorbol myristate acetate (PMA) and then exposed to human chorionic gonadotrophin (hCG) +/- isobutyl-methylxanthine (IBMX) for a further 4 h. In the absence of IBMX, cloprostenol and PMA inhibited hCG-stimulated progesterone production. However, in the presence of 0.5 mM IBMX, inhibition of phosphodiesterase prevented these antigonadotrophic effects. Phosphodiesterase activity was assessed by a novel direct assay performed on intact cells after 3 days of culture. PGF2 alpha, cloprostenol and 4 beta-PMA all enhanced cAMP degradation whilst an inactive phorbol ester (4 alpha-PMA) had no effect. Down-regulation of protein kinase C by 4 beta-PMA pre-treatment prevented the subsequent stimulation of phosphodiesterase activity by both cloprostenol and 4 beta-PMA. We conclude that the antigonadotrophic actions of PGF2 alpha in cultured human granulosa cells involve a stimulation of cAMP phosphodiesterase mediated via protein kinase C.

Mode of action of prostaglandin F2 alpha in human luteinized granulosa cells: role of protein kinase C.

It is well documented that prostaglandin F2 alpha (PGF2 alpha) inhibits progesterone production in luteal cells, but its mode of action is uncertain. It has recently been suggested that PGF2 alpha acts by activating the calcium and phospholipid-dependent protein kinase, protein kinase C (PKC). This hypothesis has been tested by comparing the site and mode of action of PGF2 alpha, a PGF2 alpha analogue (cloprostenol) and the PKC activator phorbol myristate acetate (4 beta PMA) in human granulosa-lutein cells. PGF2 alpha and cloprostenol exerted similar concentration-dependent inhibitory actions on gonadotrophin-stimulated cyclic AMP (cAMP) accumulation and progesterone production by human granulosa-lutein cells. The similarity in the actions of PGF2 alpha and cloprostenol in human granulosa-lutein cells suggests that they can be used interchangeably to study the role of PGF2 alpha in the regulation of steroidogenesis in the human ovary. Gonadotrophin-stimulated cAMP accumulation and progesterone production was also concentration-dependently inhibited by 4 beta PMA. In addition, cloprostenol and 4 beta PMA also inhibited dibutyryl cAMP-stimulated progesterone production, suggesting that these compounds inhibit LH action at sites before and after the generation of cAMP. The pre-cAMP site of action can be localised to the stimulatory G-protein (Gs) as both compounds inhibited cholera toxin-stimulated cAMP accumulation without affecting forskolin-stimulated cAMP accumulation. The post cAMP site of action can be localised to actions on cholesterol side chain cleavage enzyme, as both cloprostenol and 4 beta PMA inhibited 22R hydroxycholesterol-supported progesterone production without affecting pregnenolone-supported progesterone production. The finding that cloprostenol and 4 beta PMA interact with the steroidogenic cascade in a similar manner is indicative of a shared common mediator of their actions in human granulosa-lutein cells, i.e. PKC. The inhibitory actions of PGF2 alpha and 4 beta PMA on hLH-stimulated progesterone production were abolished in the presence of the PKC inhibitor, staurosporine. In addition, in PKC-depleted cells (achieved by exposure to 4 beta PMA for 20 h) the inhibitory actions of PGF2 alpha and 4 beta PMA were abolished. These results support the hypothesis that the inhibitory actions of PGF2 alpha are mediated by PKC in human granulosa-lutein cells.

The temporal relationship between melatonin and prolactin in women.

The relationship between the concentrations of melatonin and prolactin (PRL) over 24 hours has been investigated. Two peaks in PRL concentrations were distinguished: an evening peak (at 8:00 P.M.) and a nocturnal peak (at 6:00 A.M.). Melatonin concentrations showed a single peak (at 2:00 A.M.). In both the follicular and luteal phases of the menstrual cycle, melatonin increased 2 to 3 hours after the evening increase in PRL and 4 hours before the nocturnal rise in PRL concentrations. Oral administration of melatonin stimulated PRL release. The PRL response varied with menstrual cycle stage being significantly greater in the ovulatory (P less than 0.001) and luteal (P less than 0.01) phases than in the follicular phase. The results suggest that melatonin may be involved in control of the nocturnal PRL increase.

Direct assay for progesterone in saliva: comparison with a direct serum assay.

Direct assays have been described for the estimation of progesterone in serum and saliva. Both methods showed good inter-assay precision over a year, and the serum assay is now being used routinely in clinical laboratories. A highly significant correlation was obtained for progesterone concentrations in matched serum and saliva samples over a wide range of concentrations. The concentrations of progesterone in daily samples taken through the menstrual cycle fell within the range quoted in previous studies. The benefits of direct saliva measurement are described with reference to the long-term assessment of irregular cycles. Care in sample collection is necessary since contamination with agents such as oral cosmetics may result in overestimation of progesterone concentrations.

Changes in plasma concentrations of immunoreactive inhibin, progesterone, and bioactive gonadotrophin during pregnancy in the marmoset monkey.

This study describes the peripheral concentrations of immunoreactive (ir) inhibin, progesterone, and bioactive gonadotrophin during pregnancy in the marmoset monkey, a New World primate. Blood samples were taken every two weeks from six animals from ovulation to parturition. Plasma ir-inhibin concentrations were measured by inhibin α-subunit-directed radioimmunoassay (RIA) and a recently developed two-site immunoradiometric assay (IRMA) which is specific for inhibin αß dimer. Concentrations of α-inhibin increased (P < 0.001) during early pregnancy to reach a peak on week 12 of pregnancy and showed a positive correlation with bioassayable gonadotrophin concentrations (r = 0.5, n = 64; P < 0.001). The concentrations of both α-inhibin and gonadotrophin showed no further peaks and declined (P < 0.001) to low levels prior to birth. Concentrations of dimeric inhibin were substantially lower than those measured by RIA and did not vary significantly during pregnancy. Progesterone concentrations remained at luteal-phase levels during the first half of pregnancy and increased (P < 0.05) during the second half to reach maximum concentrations just prior to birth. The relationship between α-inhibin, gonadotrophin, and progesterone suggests that the increase in α-inhibin may be luteal in source and under the control of gonadotrophin. The absence of a second increase in α-inhibin later in pregnancy and the finding that lategestation placenta contained very little α-inhibin differs from observations in Old World primates studied and suggests that the placenta may not be a source of inhibin during pregnancy in the marmoset. The finding of high levels of α-inhibin, but not dimeric inhibin, suggests that inhibin-related molecules may have a role other than suppression of pituitary folliclestimulating hormone. © 1994 Wiley-Liss, Inc.

Circadian pattern of plasma melatonin concentrations in the marmoset monkey (Callithrix jacchus).

This study describes the concentrations of melatonin in plasma samples taken from marmoset monkeys (Callithrix jacchus) every 4 h over three 24-h periods. A circadian pattern of secretion was apparent, with higher levels recorded at night (20.00-08.00 h) than during the day (08.00-20.00 h) and a peak concentration at 20.00 h. There was a significant difference in the mean day and night concentrations (32.5 ± 4.5 pg/ml versus 49.0 ± 6.9 pg/ml, respectively) with individual concentrations ranging between<10-60 pg/ml in the day and 15-200 pg/ml at night. Circadian plasma melatonin concentrations were similar over the three 24-h periods, in male (n = 3) and female (n = 3) monkeys, and in dominant (cyclic, n = 5) and subordinate (acyclic, n = 4) females. The results show a less pronounced circadian profile in the marmoset than is seen in the human but a similar profile to that in the seasonally breeding rhesus monkey.

Stimulation of the production of progesterone by the corpus luteum of the ewe by the perfusion of melatonin in vivo and by treatment of granulosa cells with melatonin in vitro.

The ability of melatonin to stimulate luteal steroidogenesis directly in the ewe was investigated by using a perfusion cannula system in vivo and luteinised granulosa cells in vitro. Exposure to human chorionic gonodotrophin (hCG) or ovine luteinising hormone (oLH), as positive controls, significantly stimulated progesterone secretion by the perfused corpus luteum within 20 minutes and by granulosa cells after two days of culture. Melatonin perfused through the corpus luteum at a physiological concentration, significantly stimulated progesterone secretion but the response was not as rapid as that to hCG. The significant stimulation of progesterone by melatonin in the perfusion system was maintained for between 30 and 60 minutes. Melatonin significantly stimulated progesterone production by granulosa cells in vitro after two days of culture, but the degree of response depended on the size of the follicles from which the cells were obtained. These results demonstrate that melatonin can act directly on the corpus luteum to increase progesterone production, and that its mechanism of action appears to be different from that of hCG or oLH in the ewe. This action of melatonin may be related to the reported improved luteal function late in the breeding season after the prolonged exposure of ewes to melatonin.

Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro.

Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium. At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells. This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media. Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added. Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol. Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml). This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary.

Roles of cyclic AMP and inositol phosphates in the luteolytic action of cloprostenol, a prostaglandin F2 alpha analogue, in marmoset monkeys (Callithrix jacchus).

The luteolytic response to a prostaglandin F2 alpha analogue, cloprostenol, was investigated in vivo and in vitro at defined stages of the luteal phase. In vivo administration of cloprostenol to female marmoset monkeys on day 3 after ovulation had no effect on plasma progesterone concentrations, whereas administration on day 14 after ovulation reduced plasma progesterone to preovulatory concentrations within 4 h. To identify the cellular basis for this luteolytic action, marmoset luteal tissue obtained on days 3, 6 and 14 after ovulation was incubated in vitro and progesterone production, cAMP accumulation and phosphoinositide (PI) turnover measured in response to cloprostenol, human chorionic gonadotrophin (hCG) with or without cloprostenol, or dibutyryl-cAMP with or without cloprostenol. Progesterone production was stimulated by both hCG and dbcAMP at all stages of the luteal phase. Although neither hCG nor dbcAMP had any significant effects on PI turnover, hCG also increased cAMP accumulation. In marmoset luteal tissue obtained on day 3 after ovulation, cloprostenol had no significant effect on basal or hCG/dbcAMP-stimulated progesterone production but significantly stimulated PI turnover. In contrast, on days 6 and 14 after ovulation, cloprostenol significantly inhibited hCG- and dbcAMP-stimulated progesterone production and the cAMP response to hCG, but had no significant effect on PI turnover. Since progesterone production by the marmoset corpus luteum depends on the luteotrophic support of luteinizing hormone (LH), these observations suggest that the luteolytic action of cloprostenol in vivo involves the inhibition of LH/hCG action at sites both prior and subsequent to cAMP accumulation. However, such luteolytic effects do not appear to require the generation of inositol phosphates by increased PI turnover.