SUMMARY

The actions of luteinizing hormone (LH) and of prostaglandin E₂ (PGE₂) on the intact ovary or isolated components of the ovary from adult or prepubertal rats were studied in vitro with respect to (1) the rate of incorporation of [³H]adenine via ATP into cyclic adenosine-3′,5′-monophosphate (cAMP), or the level of cAMP measured by a competitive protein-binding assay; (2) protein kinase activity in the 27000 g supernatant, with calf thymus histone as the substrate; and (3) rate of oxidation of d-[6-¹⁴C]glucose. In addition, ornithine decarboxylase activity was assayed in the ovary in vitro after hormone treatment in vivo.

When ovaries from adult or pubertal rats were incubated in Krebs—Ringer bicarbonate buffer, addition of PGE₂ to the medium resulted within 1 min in a doubling of the rate of cAMP formation; the rate was about fourfold after 5 min. PGE₂ was 25 times more potent in this respect than prostaglandin F_2α. Both isolated Graafian follicles and corpora lutea responded to either PGE₂ or LH with increased cAMP production. During the perinatal period and until the age of 10 days the ovaries were unresponsive to LH, but responded to PGE₂ with increased cAMP formation. Follicles cultured with LH for 18 h and then washed were refractory to subsequent stimulation by LH, yet remained fully responsive to PGE₂.

A prostaglandin analogue, 7-oxa-13-prostynoic acid, did not inhibit LH-stimulated or PGE₂-stimulated cAMP formation in vitro in whole ovaries. Indomethacin, a substance reported to inhibit prostaglandin synthesis in other tissues, likewise failed to inhibit this action of LH. Simultaneous addition of LH and PGE₂ to the incubation medium augmented cAMP production to a significantly greater extent than did either agonist when added at maximally effective concentrations on their own, though this augmentation was short of a full additive effect. The latter finding provides presumptive evidence against the view that PGE₂ is an obligatory mediator of LH action on the ovary, but this question remains open.

PGE₂ mimicked the action of LH in stimulating glucose oxidation by ovaries in vitro, and in causing a 15-fold increase in ovarian ornithine decarboxylase activity within 4 h of injection into prepubertal rats.

Ovarian protein kinase activity in pubescent (28-day-old) rats was markedly enhanced by incubation of intact ovaries with LH or PGE₂ or by exogenous cAMP added to the 27000 g supernatant. The stimulatory action of LH or PGE₂ on the enzyme was attended by a significant decrease (to 10% of control value) in the binding of exogenous cyclic [³H]AMP to protein in the 2000 g supernatant fraction of the ovary, presumably reflecting saturation of the cAMP-binding regulatory subunit of protein kinase by enhanced endogenous generation of the cyclic nucleotide.

Stimulation of ovarian protein kinase by exogenous cAMP was demonstrable in 12-day-old rats, but insignificant at the age of 7 days. It thus appears that the competence of ovarian protein kinase to respond to cAMP, and of ovarian adenylate cyclase to respond to LH, are both acquired during the 2nd week of postnatal development.

SUMMARY

The binding of ¹²⁵I-labelled ovine luteinizing hormone (OLH) and human chorionic gonadotrophin (HCG) to unbroken cells or homogenates from rat and bovine ovaries was studied in relation to the hormonal activation of adenylate cyclase. Both hormones are preferentially bound to ovarian tissue (luteal as well as follicular), while adrenal, kidney and liver showed no specific binding. The bound HCG was associated chiefly with the fraction of the homogenate sedimenting at 1000 g . Addition of excess unlabelled hormone (HCG, OLH) together with the corresponding or heterologous ¹²⁵I-labelled hormone reduced the radioactivity bound to the tissue by 70–90%; rat LH also competed with OLH and HCG for binding sites, but follicle-stimulating hormone, prolactin, growth hormone, adrenocorticotrophin or glucagon were without effect. Binding could be dissociated from activation of adenylate cyclase by a two-stage incubation: at 4 °C rat ovarian slices bound the labelled hormones, though binding efficiency was reduced to about 20%, but no stimulation of the enzyme was observed. Reincubation of these slices, after washing, at 37 °C in hormone-free medium resulted in maximal activation of adenylate cyclase. Addition of homologous antibodies before the second incubation step abolished the effect of OLH but not that of HCG on the activation of the enzyme. Similarly, addition of homologous antibodies or of excess unlabelled hormone caused dissociation of bound ¹²⁵I-labelled OLH but not of ¹²⁵I-labelled HCG from receptor sites. It is concluded that (i) HCG, OLH and rat LH are bound to the same receptor on ovarian cells, but binding of OLH to the receptor is more labile than that of HCG; (ii) the receptor appears to be located on the plasma membrane, since it was largely associated with the 1000 g pellet and since bound OLH remained accessible to antibody; (iii) activation of adenylate cyclase requires the continued occupation of the binding site by the hormone, but occupation of only a fraction of the receptor sites by hormone is adequate to induce maximal production of cyclic AMP by the target tissue.

SUMMARY

The hypothesis that cyclic AMP plays an essential role in mediating the biological action of oestradiol on the uterus, was tested by determining the tissue concentration of the cyclic nucleotide after incubation of uteri of immature rats with oestradiol or after injection of this steroid into immature or ovariectomized rats. The effect of known stimulants of uterine adenyl cyclase, namely β-adrenergic drugs and prostaglandin E₂ (PGE₂), on the level of cyclic AMP in the uterus was also examined both in vitro and in vivo.

In either system, oestradiol failed to enhance the concentration of cyclic AMP in the uterine tissue, whereas adrenaline or the almost purely β-adrenergic agonist isoprenaline (isoproterenol) caused cyclic AMP accumulation that was susceptible to inhibition by the β-adrenergic blocking agent propranolol. Prostaglandin E₂, and to a much lesser degree prostaglandin F_2α, increased cyclic AMP concentration in the uterus, but the effect of PGE₂ was not inhibited by propranolol. It may be concluded that oestradiol does not cause appreciable stimulation of PGE₂ synthesis or activation of β-adrenergic receptors in the rat uterus since, otherwise, increased cyclic AMP production should have been observed after the treatment with oestradiol.

Isoprenaline mimicked the stimulatory action of oestradiol on uterine ornithine decarboxylase. However, this action of isoprenaline was abolished by propranolol, whereas that of oestradiol was only slightly, though significantly, inhibited.

The present findings do not support the view that the action of oestradiol on the uterus is mediated by cyclic AMP, and also suggest that β-adrenergic receptors and PGE₂ can have only a minor role, if any, in the mechanism of action of this hormone.

Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel

(Received 12 August 1975)

A specific radioimmunoassay for oestradiol was used to examine at which stage of postnatal development the ovary is capable of secreting oestrogen in response to stimulation by gonadotrophins, prostaglandin E₂(PGE₂), or the 8-bromo-derivative of cyclic AMP (8-Br-cAMP) in vitro.

Ovaries from Wistar-derived rats (6–9 days of age) were placed in incubation flasks (two ovaries per flask, at least six flasks for each experimental group) and incubated for 30 min at 37°C in 1 ml Krebs–Ringer bicarbonate buffer (KRB), pH 7·4, containing glucose (1 mg/ml) under an atmosphere of 95% O₂ : 5% CO₂. The medium was replaced by 1 ml fresh KRB, containing albumin (100 μg/ml), hormones, PGE2 or 8-Br-cAMP as indicated. The incubation was continued for another 4 h. The medium was extracted with 5 vol. of ether, and the ether extract was analysed for

Department of Biodynamics, Weizmann Institute of Science, Rehovot, Israel

(Received 4 June 1974)

Luteinizing hormone (LH) stimulates ovarian ornithine decarboxylase (ODC), the first enzyme in the pathway of polyamine synthesis, when injected into mature (Kobayashi, Kupelian & Maudsley, 1971; Jänne & Williams-Ashman, 1971) or immature rats (Kaye, Icekson, Lamprecht, Gruss, Tsafriri & Lindner, 1973). Kobayashi et al. (1971) observed a rise in ovarian ODC activity on the afternoon of prooestrus, which may be related to the ovulatory process (Kobayashi et al. 1971) and/ or the early stages of corpus luteum formation (Williams-Ashman, Jänne, Coppoc, Geroch & Schenone, 1972). We show here that ovarian ODC activity is stimulated by LH preferentially in the Graafian follicle.

Groups of three to five Wistar-derived female rats (approximately 100 days old) selected after at least two successive regular 4-day oestrous cycles, were used for surgical isolation of Graafian follicles and corpora lutea. Only corpora lutea