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Abstract
To clarify the possible mechanisms regulating prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) synthesis, the effects of gonadotropin-releasing hormone (GnRH) and substance P (SP) on the release of these two prostaglandins were studied in the oocytes of the crested newt, Triturus carnifex. Full-grown oocytes of T. carnifex, freed from follicular cells, were incubated in the presence of GnRH or SP and of the inhibitors of several enzymes involved in the release of arachidonic acid (AA) and in the conversion of AA into PGE2 and PGF2α. In parallel, the same experiments were performed on oocytes with membrane phospholipids labelled with [3H] AA. In addition, the PGE2-9-ketoreductase activity was evaluated through the conversion of [3H]PGE2 into [3H]PGF2α. The results showed that GnRH and SP could regulate prostaglandin synthesis through the activation of phospholipase C and diacylglycerol lipase, and through the modulation of PGE2-9-ketoreductase in the oocytes of T. carnifex. In particular, GnRH enhances the activty of PGE2-9-ketoreductase with a consequent increase in PGF2α, while SP inhibits the enzyme which leads to an increase in PGE2. A similar mechanism could also be hypothesized for other vertebrate species.
Journal of Endocrinology (1995) 145, 235–241
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Abstract
To clarify the endocrine mechanism involved in the short captivity stress in the water frog, Rana esculenta, the activity of 9-ketoreductase, the enzyme which converts prostaglandin E2 (PGE2) into prostaglandin F2α (PGF2α), and aromatase, which converts testosterone into oestradiol-17β, were studied. Adult male and female frogs were sacrificed 0, 1·5, 3, 6, 12, 24, 48, 72, 168 and 336 h after capture in the field. PGE2, PGF2α, progesterone, testosterone, oestradiol-17β and corticosterone plasma levels were detected by RIA at each time point. 9-Ketoreductase (conversion of [3H]PGE2 into [3H]PGF2α) and aromatase (conversion of [3H]testosterone into [3H]oestradiol-17β) activities in the brain, testis, ovary and interrenal were also determined at each time point. After capture, levels of plasma PGF2α increased (male: 228%; female: 288%) and PGE2 decreased (male: 68%; female: 81%) at 1·5 h, oestradiol-17β increased (male: 399%; female: 425%) and testosterone decreased (male: 87%; female: 83%) at 6 h, and corticosterone increased (male: 421%; female: 426%) at 72 h. 9-Ketoreductase activity in the brain was enhanced at 1·5 h after capture (male: 249%; female: 262%); aromatase activity increased at 6 h in the testis (261%), ovary (273%) and interrenal (male: 227%; female: 267%). These results indicate that short captivity stress could induce an increase in plasma PGF2α through activation of brain 9-ketoreductase. In turn, PGF2α might enhance the levels of circulating oestradiol-17β through activation of gonadal and interrenal aromatase.
Journal of Endocrinology (1996) 148, 233–239
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ABSTRACT
The effects of β-endorphin and its receptor antagonist, naloxone, on corticosterone and cortisol production in male and female Triturus carnifex were studied in vivo and in vitro. In the in-vivo experiment, the animals were injected s.c. with β-endorphin and/or naloxone, and killed after 15, 30, 90 and 360 min. In the in-vitro experiment, interrenal tissues, with and without added pituitary, were incubated with β-endorphin and/or naloxone for 15, 30, 60 and 120 min. The data obtained in vivo and in vitro from males and females were in agreement. Treatment with β-endorphin caused a significant decrease in corticoster-one and cortisol release, while naloxone induced an increase in the two corticosteroids at the same times as the decrease caused by β-endorphin. The combined β-endorphin plus naloxone treatment did not change corticosterone and cortisol levels. These results suggest that, in Triturus carnifex, opioids are involved in the regulation of the hypothalamo-pituitary-interrenal axis. In particular, the in-vitro results indicate a direct effect of opioids on interrenal steroidogenesis.
Journal of Endocrinology (1991) 131, 295–302
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By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2alpha (PGF-2alpha) induced in the corpora lutea (CL) of pseudopregnant rabbits, we have demonstrated that these prostaglandins modulate luteal nitric oxide synthase (NOS) activity and progesterone production differently, depending on the age of the CL. On CL obtained on day 4 of pseudopregnancy (day-4), PGE-2 was found to depress NOS total activity to 13% of control and to significantly increase basal progesterone secretion by 61%, while PGF-2alpha had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2alpha caused a 2.5-fold increase of NOS activity and a marked decrease in progesterone production. Using specific inhibitors, we found that the regulatory actions of PGE-2 in vitro are mediated via the adenyl cyclase/protein kinase A (PKA) second messenger system, while the PGF-2alpha-induced luteolytic effects on day-9 CL depend upon activation of the phospholipase C/protein kinase C (PKC) system. The different responsiveness of day-4 and day-9 CL to PGE-2 and PGF-2alpha could depend on receptor availability for these two prostaglandins, even if other cellular mechanisms cannot be excluded. The present study supports a functional role for NOS in regulating the steroidogenic capacity of rabbit CL, and reveals a novel interaction between a stimulatory G-protein-coupled receptor and PKC/PKA-mediated signal transduction modulating NOS activity.
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We examined the presence and the regulation of nitric oxide (NO) synthase (NOS) using in vitro cultured corpora lutea (CL) obtained from rabbits at days 4 and 9 of pseudopregnancy. The role of NO and NOS on steroidogenesis was also investigated using the same CL preparations after short-term incubations (30 min and 2 h) with the NO donor, sodium nitroprusside (NP), the NOS inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME) and prostaglandin (PG) F-2alpha. The basal NOS activity was greater in CL at day 4 than at day 9, and was also differently modulated by PGF-2alpha, depending on the age of the CL. The addition of PGF-2alpha to day 4 CL had no effect, but PGF-2alpha on day 9 caused a threefold increase in NOS activity. NP caused a two- to fivefold decrease in release of progesterone from CL of both ages, and this inhibitory effect on steroidogenesis was reversed by l-NAME. All treatments failed to modify basal androgens and 17beta-oestradiol was not detectable in either control or treated CL. These results suggest that NO is effectively involved in the regulation process of steroidogenesis, independently of 17beta-oestradiol. PGF-2alpha had no effect on day 4, but induced luteolysis on day 9, by reducing progesterone (P</=0. 01) to about 18% of control. The luteolytic action of PGF-2alpha was completely reversed by co-incubation with l-NAME, thus supporting the hypothesis that luteolysis is mediated by NO. The addition of NP or l-NAME did not modify the in vitro release of PGF-2alpha. We hypothesised that PGF-2alpha upregulates NOS activity and, consequently, the production of NO, which acutely inhibits progesterone release from day 9 CL of pseudopregnant rabbits.
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Studies were conducted to characterize receptors for prostaglandin (PG) F(2alpha) (PGF(2alpha)) and PGE(2), and the signalling pathways regulating total nitric oxide synthase activity and progesterone production in rabbit corpora lutea (CL) of different luteal stages. CL were obtained at days 4, 9 and 13 of pseudopregnancy and cultured in vitro for 2 h with PGF(2alpha) or PGE(2) and with activators and inhibitors of G protein (Gp), phospholipase C (PLC), protein kinase C (PKC), adenylate cyclase (AC) and protein kinase A (PKA). High affinity PGF(2alpha) receptor (K(d)=1.9+/-0.6 nM mean+/-s.e.m. ) concentrations increased (P< or =0.01) four- to five-fold from early to mid- and late-luteal phases (50.6+/-8.5, 188.3+/-36.1 and 231.4+/-38.8 fmol/mg protein respectively). By contrast, PGE(2) receptor (K(d)=1.6+/-0.5 nM) concentrations decreased (P< or =0.01) from day 4 to day 9 and 13 (27.5+/-7.7, 12.4+/-2.4 and 16.5+/-3.0 fmol/mg protein respectively). The Gp-dependent AC/PKA pathway was triggered only on day 4 CL, mimicking the PGE(2) treatment and increasing progesterone production. In both day 9 and day 13 CL, the Gp-activated PLC/PKC pathway evoked a luteolytic effect similar to that induced by PGF(2alpha). The time-dependent selective resistance to PGF(2alpha) and PGE(2) by rabbit CL is mediated by factors other than a lack of luteal receptor-ligand interactions.
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Abstract
The presence of pituitary adenylate cyclase-activating peptide (PACAP) 38-immuno-like material (PACAP 38-IL) in the brain and ovary of the crested newt, Triturus carnifex, and its action on ovarian steroidogenesis and prostaglandin synthesis were evaluated. The HPLC brain and ovary extract peaks that eluted like PACAP 38 were considered PACAP 38-like material. The concentrations of PACAP 38-IL in the HPLC extracts were measured by RIA. T. carnifex ovary was incubated with PACAP 38, brain and ovary PACAP 38-IL, and inhibitors of cyclooxygenase (COX), adenylate cyclase (AC) and phospholipase C (PLC) for 30 and 60 min. PACAP 38, and brain and ovary PACAP 38-IL increased prostaglandin E2 (PGE2) (30 and 60 min), and progesterone and corticosterone (60 min), but decreased oestradiol-17β (60 min). COX and PLC inhibitors counteracted the increases in PGE2, progesterone and corticosterone and the decrease in oestradiol-17β, and the AC inhibitor also counteracted them except for PGE2. These results suggest that PACAP 38-IL, present in T. carnifex brain and ovary, acts on PLC, inducing the increase of PGE2 which, in turn, acting on AC, induces increases in progesterone and corticosterone and a decrease in oestradiol-17β.
Journal of Endocrinology (1997) 152, 141–146
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Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.
Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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Dipartimento di Scienze Biopatologiche Veterinarie, Laboratorio di Biotecnologie Fisiologiche, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
Dipartimento di Scienze Biopatologiche Veterinarie, Sezione di Anatomia, Università di Perugia, S Costanzo 4, 06100 Perugia, Italy
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In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2α and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2α, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2α release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2α production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.
