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By examining in vitro the effects of prostaglandin E-2 (PGE-2) and prostaglandin F-2 (PGF-2 ) 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-2 had no effect. On day-9 CL, PGE-2 was ineffective, but PGF-2 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-2 -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-2 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.

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2a synthesis following PGF2a treatment at either early-(day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P!0.01) 4-to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P!0.01) COX-2 and PGE2-9-K basal activities, and PGF2a synthesis rate, but higher (P!0.01) PGE2 production. Independent of luteal stage, PGF2a treatment did not affect COX-1 activity. In day-4 CL, PGF2a induced an increase (P!0.01) in both COX-2 activity and PGF2a synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2a up-regulated (P!0.01) both COX-2 and PGE-9-K activities, and PGF2a production, but decreased (P!0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2a challenge and were more marked in day-9 CL. Our data suggest that PGF2a directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2a synthesis in an autoamplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2a in rabbits.

The aim of this study was to better understand the role of the endothelin-1 (ET-1) system in the process of controlling the corpora lutea (CL) life span in rabbits. ET-1 (10 microg iv) administration at d 9 and 12 of pseudopregnancy induced a functional luteolysis within 24 h of injection, but it was ineffective at both d 4 and 6. Pretreatments with Bosentan, a dual ET(A)/ET(B) receptor antagonist, or cyclooxygenase (COX) inhibitor blocked the luteolytic action of ET-1 but not that induced by prostaglandin F2alpha (PGF2alpha). In CL cultured in vitro, ET-1 increased (P

Total activity of nitric oxide synthase (NOS) and the gene expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) isoforms in corpora lutea of pseudopregnant rabbits were examined during prostaglandin F 2␣ (PGF 2␣ )-induced luteolysis. Corpora lutea were collected at 0, 6, 12, 24 and 48 h after an injection of PGF 2␣ at day 9 of pseudopregnancy. At 12 h after PGF 2␣ administration, luteal mRNA encoding eNOS decreased (P 0.05) by 40% and remained low throughout the subsequent 36 h, whereas eNOS protein increased (P 0.05) two-to threefold. By contrast, expression of mRNA encoding iNOS was poor and remained fairly constant, but transcription increased eightfold (P 0.01) within 6 h after PGF 2␣ treatment and then decreased to values similar to those of controls. Total NOS activity increased twofold (P 0.01) at 6 h after treatment and remained high thereafter, whereas progesterone concentrations in explanted corpora lutea decreased (P 0.01) from 302.4 ± 42.3 pg mg −1 at day 9 to 58.6 ± 8.3 at 48 h later, and peripheral plasma concentrations of progesterone declined too. Long-term administration of N -nitro-L-arginine methyl ester (0.6 g l −1 per os) from day 2 of pseudopregnancy onward partially blocked the luteolytic action of PGF 2␣ administered at day 9 of pseudopregnancy. In nitric oxide (NO)-deficient rabbits, progesterone concentrations remained higher (P 0.01) than in controls at 24-48 h after PGF 2␣ administration (4.5 to 3.2 ng ml −1 , respectively). These data are the first to characterize NOS activity. The time course of expression of eNOS and iNOS in rabbit corpora lutea during PGF 2␣ -induced luteolysis gives additional support to a physiological role of NO in the regulation of regression of corpora lutea in rabbits.

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, N -nitro--arginine methyl ester (-NAME) and prostaglandin (PG) F-2 . The basal NOS activity was greater in CL at day 4 than at day 9, and was also differently modulated by PGF-2 , depending on the age of the CL. The addition of PGF-2 to day 4 CL had no effect, but PGF-2 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 -NAME. All treatments failed to modify basal androgens and 17 -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 17 -oestradiol. PGF-2 had no effect on day 4, but induced luteolysis on day 9, by reducing progesterone (Pc0·01) to about 18% of control. The luteolytic action of PGF-2 was completely reversed by co-incubation with -NAME, thus supporting the hypothesis that luteolysis is mediated by NO. The addition of NP or -NAME did not modify the in vitro release of PGF-2 . We hypothesised that PGF-2 upregulates NOS activity and, consequently, the production of NO, which acutely inhibits progesterone release from day 9 CL of pseudopregnant rabbits.

We studied leptin involvement in rabbit corpora lutea (CL) activity, and its post-transcriptional signalling pathway. The expression of leptin receptor (Ob-R) in rabbit ovary at day 9 of pseudopregnancy was evaluated by immunohistochemistry and Western blot analysis. The specificity of the Ob-R receptor antibodies was characterised by immunoprecipitation and competition with blocking peptide. Day 9 CL were incubated in vitro with leptin alone or with inhibitors of PLC (phospholipase C), PLD (phospholipase D), AC (adenylate cyclase), JAK (janus kinase), MAPK (mitogen-activated protein kinase) and both cAMP-and cGMP-specific PDE (phosphodiesterase). Prostaglandin F2 (PGF2 ), PGE2 and progesterone levels were measured in the culture medium, while NOS (nitric oxide synthase) and cAMP-and cGMP-specific PDE activities were measured in CL tissue. Positive staining for Ob-R was found within the cytoplasm of large luteal cells of CL as well as in granulosa cells of follicles and oocytes. Immunoblots detected a band of about 99 kDa size in Ob-R immunoprecipitates from CL homogenates. This band was not detectable after pre-incubation of the primary antibody with the immunising leptin peptide. Leptin increased PGF2 and cAMPspecific PDE, decreased basal progesterone and did not affect PGE2 and NOS levels. Leptin used the JAK pathway in increasing PGF2 , and MAPK and cAMPspecific PDE in decreasing progesterone. This study supports a permissive luteolytic role for leptin in rabbit CL.

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