Relationship between endothelin 1 and nitric oxide system in the corpus luteum regression

doi:10.1016/j.plefa.2003.07.002

Prostaglandins, Leukotrienes and Essential Fatty Acids

Volume 69, Issue 5, November 2003, Pages 359-364

https://doi.org/10.1016/j.plefa.2003.07.002 Get rights and content

Abstract

The present study was designed to investigate the relationship between the nitric oxide (NO) system and endothelin 1 (ET-1) in the mechanism of corpus luteum (CL) development and consequently regression in rats. We first evaluated basal ET-1 levels in ovarian tissue from rats with different stages of CL development. An increased ovarian ET-1 content was found during CL regression. In a dose-department response, ET-1 decreased progesterone (P4) and increased prostaglandin (PG) PGF2α production. By means of a competitive nitric oxide synthase (NOS) inhibitor: L-nitro arginine methyl ester (L-NAME) and a slow NO releasing: diethyl-aminetriamine (DETA-NONOate), we demonstrated that NO system could be the intermediary in the ET-1 diminishing P4 production. The Western blot analysis revealed an increase on iNOS while eNOS protein expression was diminished. We also found a diminution of total NOS activity after ET-1 treatment. These data suggest the existence of a functional relationship between ET-1 and NOS isoforms leading the regulation of CL functionally.

Introduction

The life span and function of corpus luteum (CL) are controlled by both stimulatory (luteotropic) and inhibitory (luteolytic) factors. Endothelin-1 (ET-1), a potent vasoconstrictor factor originally isolated from porcine vascular endothelial cells [1] is a 21-amino acid peptide member of a structurally homologous peptide family, which includes ET-2 and ET-3 [2]. Although ETs are products of three different genes, they share extensive sequence homology and a common structural design [3]. Within the ovary, high concentrations of ET-1 were found in the human follicular fluid [4], [5]. Furthermore, increased concentrations of ET-1 mRNA were detected in rat and bovine corpus luteum [6], [7] and in human granulosa cells [5].

Moreover, the involvement of endothelial cells in CL physiology is supported by their abundance (>50% of total CL cells [8]) and their contact association with steroidogenic cells [9]. Recently, a novel paracrine/autocrine regulation of human luteal function involving prostaglandins (PGs), progesterone (P4) and ETs has been reported [10], [11], [12] however, the specific mechanism remains uncertain.

Nitric oxide (NO) is an endogenous mediator of numerous physiological processes, including vascular tone, platelet function, neurotransmission, host defence mechanism [13], ovarian function [14], [15] and embryonic development [16]. Formation of NO from l-arginine is catalyzed by a diverse family of nitric oxide synthase (NOS) isoenzymes [17]. The isoforms were divided into two functional classes on the basis of their dependence on calcium for their enzymatic activity i.e., the inducible isoform (iNOS) with calcium independent activity and neuronal and endothelial synthase dependent on calcium (nNOS and eNOS, respectively) [18].

In previous studies on the mechanism of CL regression, we reported that during luteolytic process, NO produced locally showed an antisteroidogenic effect, diminishing P4 levels and increasing PGF2α production [19]. We also found that during luteolysis, both NO and PGF2α are involved in the regulation of oxidative parameters [20]. On the other hand, we found that during mid-luteal phase NO has a protective role resulting in an increase of P4 production [21].

Considering the above data, the relationship between NO system-ET-1 established in other systems and the important contribution of endothelial cells in CL regression, we decided to study if the regulation of P4 and PGF2α by ET-1 during the CL regression could be mediated by the NO system and which specific NOS-isoform could be involved.

Section snippets

Animals

The animal model used was the same as that described previously [22]. Briefly, immature (28–30 days) female rats of the Wistar strain were given 15 IU/rat of pregnant mare's serum gonadotropin (PMSG; Sigma Chemical Co, St. Louis, MO, USA). We considered day 0 of psp at 48 h post-injection (ovulation). The rats were housed under conditions of controlled temperature (22°C) and illumination (14 h light; 10 h dark; lights on at 05:00) and allowed free access to Purina rat chow and water ad libitum.

Ovarian ET-1 content

Basal ET-1 production from ovarian tissue of different stages of CL development was quantified by specific EIA kit. As can be seen in Fig. 1, ovarian tissue from rats at mid stage of psp (day 5) showed lower content of ET-1 than those from rats on later stage of psp (day 8 of psp, ^*P<0.05). On day 9 ET-1 values returned to those found at 5th day.

Effect of ET-1 on progesterone and PGF2α production

In order to understand the effect of ET-1 on P4 and PGF2α production, ovaries from rats at mid-stage of psp were incubated with different doses (10⁻⁷,

Discussion

The CL is an endocrine temporary gland, which in response to LH produces P4. Very little is known regarding the behavior of the great variety of mediators responsible for differentiation and functionality of CL. Nevertheless, there is evidence that suggests that ET-1 acts as a local modulator in reproductive physiology. The abundance (more than 50% of the total cells of CL) and close association to steroidogenic cells, made scientists suspect that endothelial cells and ET-1 could have an

Acknowledgements

Authors would like to thank Maria Ines Casella and Ramona Morales for their technical support and Silvia Zorz and Curtis Pokrant for reviewing this manuscript.

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Endothelin-1 (ET-1) has been reported to mediate prostaglandin (PG) F₂α (PGF₂α)-induced luteolysis. Prostaglandins E (PGE; PGE₁ + PGE₂) are associated with implantation, maternal recognition of pregnancy, and are antiluteolytic and luteotropic in vitro and in vivo. ET-1 increased PGE secretion by bovine luteal tissue in vitro from cows where estrus was not synchronized or when estrus was synchronized with lutalyse and did not affect luteal PGF₂α or progesterone secretion, which does not support the concept that ET-1 is luteolytic or mediates PGF₂α luteolysis. Therefore, the objective of this experiment was to determine whether ET-1 infused every 6 h from 2400 h on day 10–1800 h on day 18 of the ovine estrous cycle either into the interstitial tissue of the ovarian vascular pedicle (IP) or intrauterine (IU) adjacent to the luteal-containing ovary was luteolytic in ewes. Treatments were: Vehicle-IP; Vehicle-IU; ET-1-IP; or ET-1-IU. Weights of corpora lutea differed (P ≤ 0.05) among treatment groups. Weights of corpora lutea at 1800 h on day 18 were: VEH-IP—247 ± 38 mg; VEH-IU—195 ± 31 mg; ET-1-IP—626 ± 74 mg; and ET-1-IU—542 ± 69 mg. Luteal weights on day 18 in ET-1-IP or ET-1-IU-treated ewes did not differ (P ≥ 0.05), but were heavier (P ≤ 0.05) than in the Vehicle-IP or Vehicle-IU treatment groups which did not differ (P ≥ 0.05). Profiles of progesterone in jugular venous plasma of both control groups treated with Vehicle-IP or Vehicle-IU were lower (P ≤ 0.05) than in ewes treated with ET-1-IP or ET-1-IU, which did not differ (P ≥ 0.05) between ET-1-IP or ET-1-IU treatment groups. Treatment with ET-1-IP or ET-1-IU increased (P ≤ 0.05) the PGE:PGF₂α ratio when compared to the Vehicle-IP or Vehicle-IU treatment groups, which did not differ (P ≥ 0.05) between each other. In summary, ET-1 prevented the decrease in luteal weights and the decline in progesterone, but increased the PGE:PGF₂α ratio when compared to controls. Therefore, it is concluded that ET-1 is not luteolytic in ewes, but instead may be luteotropic or antiluteolytic by altering uterine secretion of the PGE:PGF₂α ratio, since PGE₁ or PGE₂ are luteotropic in vitro and in vivo, PGE₁ or PGE₂ prevent PGF₂α-induced luteolysis in vitro and in vivo, and PGE₁ and PGE₂ increase two-fold in ewe endometrium to prevent luteolysis during early pregnancy.
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Citation Excerpt :
It is therefore likely that luteal phase defect is caused by the altered regulation of luteal blood flow during the midluteal phase. Vasoactive substances such as nitric oxide, endothelin, or angiotensin have been reported to be involved in luteal function (30–34). Further studies are needed to elucidate the relationship between luteal phase defect, luteal blood flow, and vasoactive substances.
To examine changes in blood flow in the corpus luteum throughout the luteal phase and during early pregnancy.
Longitudinal and cross-sectional prospective studies.
University hospital and city general hospital.
Sixty-one women with normal menstrual cycles and normal luteal function, 13 women with hCG-induced ovulatory cycle, 10 women with luteal phase defect, six women with luteinized unruptured follicle (LUF), and 17 pregnant women (4–10 weeks of gestation).
Blood-flow impedance in the corpus luteum was assessed by transvaginal color-pulsed Doppler ultrasound.
Resistance index (RI) in the corpus luteum.
In the normal menstrual cycle, the RI of the preovulatory follicle was high and significantly decreased after ovulation. Luteal-RI further decreased during the early to midluteal phase but significantly increased during the late luteal phase. Those changes in luteal-RI were similar to those of the hCG-induced ovulatory cycle. Luteal-RI during the midluteal phase was significantly higher in the patients with luteal phase defect than in women with normal luteal function. Luteal-RI of the LUF patients remained high throughout the luteal phase. In pregnant women, luteal-RI remained at the midluteal phase level until 7 weeks of gestation and significantly increased thereafter.
The change in luteal-RI was associated with corpus luteum development and corpus luteum regression. Luteal-RI was closely associated with luteal function.
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The aim of these studies was to investigate the effect of LH, progesterone (P4), PGE, noradrenaline (NA) and a nitric oxide donor, S-nitroso-N-acetylpenicillamine (S-NAP), on steroid acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD) and cytochrome P450 side chain cleavage (P450scc) gene expression and on the synthesis of their protein products. Bovine luteal cells were collected and prepared on days 6–10 of the estrous cycle and preincubated in vitro for 24 h. Thereafter, medium was changed and supplemented with one of six treatments: control medium, LH (100 ng/ml), P4 (10⁻⁵ M), PGE2 (10⁻⁶ M), NA (10⁻⁵ M) or S-NAP (10⁻⁴ M). In Experiment 1, luteal cells (10⁶/well) were incubated for 3, 6, 18 and 24 h. After incubation, total RNA was isolated and P4 concentrations in medium was determined. Semiquantitative RT-PCR was used to measure gene expression. In Experiment 2, luteal cells were preincubated for 24 h, then stimulated as in Experiment 1. Total protein was isolated from lysed cells and Western blot analysis was performed using specific antibodies against the StAR, 3β-HSD and cytochrome P450scc proteins. Bands were analyzed by means of KODAK 1D Image Analysis Software. In Experiment 1, LH and PGE2 stimulated secretion of progesterone from luteal cells. Concentrations of mRNA for StAR, 3β-HSD, cytochrome P450scc were increased after 6 h in cells stimulated with LH, PGE2 and P4 (P < 0.05). Gene expression was not affected by NA. In Experiment 2, LH, P4 and PGE2 induced an increase in the concentration of these three proteins. S-NAP inhibited both concentrations of mRNA and protein for StAR, 3β-HSD, cytochrome P450scc. Therefore, the increase in secretion of P4 induced by LH and PGE2 is associated with increases in StAR, 3β-HSD and cytochrome P450scc gene expression. This genomic response may be mediated in part through a positive effect of P4 on the expression of these genes observed in this experiment.
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A dense network of capillaries irrigates the corpus luteum (CL) allowing an intricate cross talk between luteal steroiodgenic and endothelial cell (EC) types. Indeed, luteal endothelial cells (LEC) play pivotal roles throughout the entire CL life-span. Microvascular endothelial cells are locally specialized to accommodate the needs of individual tissues, therefore unraveling the characteristics of LEC is imperative in CL physiology. Numerous studies demonstrated that endothelium-derived endothelin-1 (ET-1) is upregulated by the luteolytic hormone-prostaglandin F2α (PGF2α) and functions as an important element of the luteolytic cascade. To have a better insight on its synthesis and action, members of ET system (ET-1, ET converting enzyme -ECE-1 and ET_A and ET_B receptors) were quantified in LEC. The characteristic phenotype of these cells, identified by high ET-1 receptor expression (both ET_A, ET_B) and low ET-1 and ECE-1 levels, was gradually lost during culture suggesting that luteal microenvironment sustains the selective phenotype of its resident endothelial cells. Proper vascularization and endothelial cell activity per se are essential for normal CL function. Therefore, factors affecting vascular growth are expected to play major role in the regulation of luteal function. Concomitantly with the angiogenic process, luteal PGF2α and its receptors (PGFR) are induced and maintained during most of the CL life-span, suggesting a possible role of PGF2α in LEC proliferation and function. Dispersed LEC expressed PGFR and incubation with the prostaglandin stimulated mitogen-activated protein kinase (MAPK) signaling cascade. PGF2α activated p42/44 MAPK phosphorylation also in long-term cultured LEC. In this cell type, PGF2α increased cell number, ³H-Thymidine incorporation and cell survival. Additionally, PGF2α rapidly and transiently stimulated the expression of immediate-early response genes, i.e. c-fos and c-jun mRNA, further suggesting a mitogenic effect for this prostaglandin in LEC. These data imply that PGF2α may assume different and perhaps opposing roles depending on luteal microenvironment.
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^☆: Supported by Programa Latinoamericano de Capacitación e Investigación en Reproducción Humana Re-entry Grant PRE-049–2001.

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Published by Elsevier Ltd.

Relationship between endothelin 1 and nitric oxide system in the corpus luteum regression☆

Abstract

Introduction

Section snippets

Animals

Ovarian ET-1 content

Effect of ET-1 on progesterone and PGF2α production

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Pharmacol. Ther.

Biochem. Pharmacol.

Anal. Biochem.

FEBS Lett.

Mol. Cell. Endocrinol.

J. Biol. Chem.

A novel potent vasoconstrictor peptide produced by vascular endothelial cells

Nature

The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes

Proc. Natl. Acad. Sci. USA.

Activation of multiple mechanisms including phospholipase D by endothelin-1 in rat aorta

Am. J. Physiol.

Anovel potent vasoconstrictor peptide produced by vascular endothelial cells

Nature

Regulation of endothelin-1 expression in the bovine corpus luteum: elevation by prostaglandin F2α

Endocrinology

Morphometric analysis of the cellular composition of the ovine corpus luteum

J. Anat.

Vascular development and heparin-binding growth factors in the bovine corpus luteum at several stages of the estrous cycle

Biol. Reprod.

Inter-relationships between endothelin and prostaglandin F2α in corpus luteum function

Rev. Reprod.

Endothelin-1 mediates prostaglandin F2α-induced luteal regression in the ewe

Biol. Reprod.

Endothelins enhance prostaglandin (PGE2 and PGF2α) biosynthesis and release by human luteal cells: evidence of a new paracrine/autocrine regulation of luteal function

J. Clin. Endocrinol. Metab.