Department of Neuroendocrinology, Medical Center of Postgraduate Education, 04158 Warsaw, Poland
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Abstract |
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Key words: endothelin/follicular fluid/oestradiol/progesterone/testosterone
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Introduction |
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The cleavage of mature 21 residue ET from 38 amino acids precursors (`big ET') is performed intracellularly by specific ET generating enzyme (Ohnaka et al., 1992). However, considerable amounts of big ET pass through the cell membrane and are present in circulation and in follicular fluid (FF) as well (Miauchi et al., 1989
; Haq et al., 1996
). Although the molar ratio of ET-1:ET-2:big ET-1 in FF has been reported to be approximately 1:2:1 (Haq et al., 1996
), only the 21 amino acid ET-1 and ET-2 are the active forms which may play a role as intra-ovarian factors regulating the steroidogenesis. In the present study, using a radioimmunoassay specific for only 21 residue ET, the total concentration of the active ET was evaluated in FF in relation to follicle development and steroidogenesis in women following gonadotrophin stimulation for in-vitro fertilization (IVF) protocols.
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Materials and methods |
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Radioimmunoassay for 21 amino acid ET
The concentrations of ET were determined by radioimmunoassay using a commercially available kit (Amersham-Pharmacia, Little Chalfont, Bucks, UK; RPA 5559) after prior extraction of the peptide from FF using Amprep 50 mg C2 columns (Amersham-Pharmacia). In this assay synthetic 125I-ET-3 was used as a tracer. The primary antibody (rabbit) recognized common C-terminal structure of the 21 residue ET, and therefore did not crossreact with big ET. The crossreactivity with ET-1, ET-2, ET-3, and big ET-1, determined by concentration giving 50% B/B0 (tracer binding), was 100, 144, 52 and 0.4% respectively. Values are mean of two parallel measurements of each FF sample. The intra- and inter-assay coefficient of variation was 4.8 and 13.8% respectively. The method sensitivity was 3.6 pmol/l, as given by the manufacturer.
Hormone measurements
Oestradiol, progesterone, and testosterone concentrations in FF were measured in duplicate after 1:100 dilution in serum-free assay buffer (sodium phosphate 0.1 mmol/l + NaCl 0.3 mol/l + sodium azide 0.9 g/l, pH 7.5) using the following commercially available kits: oestradiol and progesterone enzyme immunoassay (EIA) (bioMerieux, Lyon, France); testosterone radioimmunoassay (Orion Diagnostica, Espoo, Finland). Serum concentrations of luteinizing hormone (LH), oestradiol and progesterone on the day of the oocyte retrieval were measured using EIA kits (bioMerieux).
Statistical analysis
Normality of measurable variables distribution was checked with KolmogorowSmirnov test, and Spearman rank order correlation non-parametric test was used for analysis of the data. P < 0.05 was considered significant.
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Results |
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Discussion |
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The concentration of ET in FF correlated negatively with follicle diameter, which suggests that synthesis of ET within the ovary may depend upon the stage of follicle maturation. The results presented here are consistent with the study of Kamada (Kamada et al., 1993), in which it was reported that ET-1-like immunoreactivity in fluid obtained from human immature follicles was significantly higher than that in fluid from the mature and post-mature follicles. Animal and human granulosa cells were shown to express mRNA for prepro-ET-1 in vitro, and therefore appear to be the most likely source of ET present in FF (Kubota et al., 1994
; Magini et al., 1996
). One of the factors that may up-regulate ET gene expression in the follicular environment during the early stage of growth appears to be the low oxygen tension in FF (Gosden and Byatt-Smith, 1986
). Hypoxia was identified as a potent stimulus for ET-1 gene expression in the endothelial cells of peripheral blood vessels (Kourembanas et al., 1991
). Because ET-1 and ET-3 are potent mitogens for human vessels (Nausee et al., 1990
), a high concentration of ET may promote angiogenesis in premature follicles. Vascularization of the growing follicle results in an increase in oxygen saturation of FF and, consequently, in reduction of ET synthesis. Thus, ET may contribute to a local negative feedback mechanism controlling the development of follicle vasculature. The significance of ET to follicle physiology may also include an auto/paracrine stimulation of DNA synthesis in granulosa cells (Kubota et al., 1994
). It is noteworthy that ET-1 concentration in FF and in peripheral plasma increases upon gonadotrophin stimulation (Magini et al., 1996
). Although the concentration of ET in FF declines during follicle development, the absolute amount of active ET in the mature follicle is about 56 times higher then that in follicles of
15 mm diameter (Kamada et al. 1993
). This amount represents considerable pool of ET that may penetrate, along with other vasoactive substances of ovarian origin, to the peripheral circulation and contribute to the development of clinical signs of the ovarian hyperstimulation syndrome (reviewed by Elchalal and Schenker, 1997
).
The significant negative correlation between ET and progesterone concentration in FF found in this study corresponds with previous in-vitro reports on prevention of luteinization by ET, and suggests that similar regulation also takes place in human ovaries in vivo. Low amounts of ET were demonstrated to suppress in a dose-dependent manner LH-stimulated accumulation of progesterone in cultured porcine granulosa cells (Iwai et al., 1991). The subsequent studies indicated that ET-1 mediated inhibition of follicle stimulating hormone (FSH)-stimulated progesterone production represents the combined ability of ET-1 to stimulate key step enzymes in progesterone biosynthesis, e.g. cholesterol side-chain cleavage (CSCC) enzyme and 3
-hydroxysteroid dehydrogenase/isomerase, and to stimulate the activity of the progesterone-degrading enzymes 20
-hydroxysteroid dehydrogenase, 5
-reductase and 3
-hydroxysteroid dehydrogenase (Tedeschi et al., 1992
). The most important action of ET in regulation of steroidogenesis appears to be the inhibition of CSCC, which is a rate-limiting enzyme. The cellular mechanism whereby ET may interfere with gonadal steroid biosynthesis seems to be related to inhibition of cyclic AMP accumulation (Furger et al., 1995
). Indeed, FSH-induced activity of both, CSCC and 3
-hydroxysteroid dehydrogenase/isomerase depends on cAMP concentration. On the other hand, accumulation of cAMP in cultured granulosa cells in response to LH stimulation was significantly decreased by co-treatment with ET-1 (Kubota et al., 1994
). A recent report (Apa et al., 1998
) indicates that inhibitory effect of ET-1 on basal and HCG-stimulated progesterone secretion from human luteal cells is exerted through ETA receptors and the protein kinase C pathway.
In contrast to in-vitro results, clinical findings failed to show a significant relationship between ET and ovarian steroids in a series of pooled samples of human FF (Kamada et al., 1993; Abae et al., 1994
; Haq et al., 1996
). It is possible that due to high between-subject variation of data the analysis of all samples in one array could have led to the loss of statistical significance. Therefore, in the present study, the correlation coefficients were evaluated separately for each patient from whom three or more specimens of FF were collected. Using this method, a highly significant relationship was found between active ET and progesterone in five of seven women. The same proportion of patients showed also marked correlation between ET and testosterone. The meaning of the latter finding is unclear, however, testosterone was shown to increase ET-1 concentration in peripheral blood. Significant sexual dimorphism in circulating plasma ET-1 concentrations has been found (Polderman et al., 1993
). Those authors reported also that testosterone treatment of female-to-male transsexuals resulted in marked elevation of plasma ET-1 concentration. Thus, it can be postulated that testosterone of stromal origin can up-regulate the expression of ET genes in granulosa cells.
It was not possible to determine any uniform pattern of relationship between ET and follicular oestradiol concentration. A similar lack of correlation was demonstrated in previous clinical reports (Kamada et al., 1993; Abae, 1994; Haq et al., 1996
). In-vitro experiments provide conflicting data on the effect of ET on oestradiol secretion. An earlier study reported a significant increase in oestradiol production after both, incubation and perfusion of rat pre-ovulatory follicles (Usuki et al., 1991
). An increase in oestradiol and progesterone secretion after short term (2 h) incubation was noted in another study (Kubota et al., 1994
) in human ovaries; however, those authors observed significant inhibition of FSH- and HCG-stimulated accumulation of progesterone during 48 h incubation. Long term exposure to ET-1 and ET-3 led also to the inhibition of oestrogen and cAMP production in rat granulosa cells (Calogero et al., 1998
). On the other hand, oestradiol treatment was found to decrease ET-1 concentration in peripheral blood (Polderman et al., 1993
), but had no influence on specific ET-1 binding by rat granulosa cells (Otani et al., 1996
). The significance of the mutual influence of ET and oestradiol on the physiology of human ovary requires further studies. Such a study assessing number of oocytes, fertilization and cleavage, with a larger sample size, is currently underway.
In conclusion, the correlation between active ET and gonadal steroids in human FF observed in the present study support the evidence that this group of peptides plays an important role in the development of ovarian follicles, especially in the inhibition of premature luteinization of granulosa cells.
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Notes |
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References |
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Submitted on February 9, 1999; accepted on June 16, 1999.