Human Reproduction, Vol. 14, No. 9, 2323-2327,
September 1999
© 1999 European Society of Human Reproduction and Embryology
Correlation between 21 amino acid endothelin, intrafollicular steroids and follicle size in stimulated cycles
Department of Neuroendocrinology, Medical Center of Postgraduate Education, 04–158 Warsaw, Poland
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Abstract |
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Several in-vitro studies have shown that endothelins (ET) may inhibit synthesis of progesterone and prevent luteinization of granulosa cells. In the present study, a specific radioimmunoassay was used to evaluate the correlation between concentrations of active (21 residue) ET and ovarian steroids in 47 samples of human follicular fluid (FF) following gonadotrophin stimulation for in-vitro fertilization (IVF) protocols. An isoform non-selective antibody was used in the radioimmunoassay, which recognized the C-terminal structure of the 21 residue ET, and therefore did not crossreact with their weakly active precursors – big ET. In pooled samples of follicular fluid (FF), the concentration of 21 amino acid ET correlated negatively with diameter of the follicles (r = –0.31, P < 0.05) and progesterone concentrations in FF (r = –0.56, P < 0.001). A positive relationship (non-significant) was found between ET and testosterone concentrations. No correlation between ET and oestradiol was observed. The within-patient correlation coefficients were also evaluated in women from whom three or more samples of FF were obtained. ET were markedly inversely correlated with follicle size in all cases, and with progesterone in five of seven women. Five of seven patients also showed significant positive correlation of ET with testosterone. The results demonstrate clinical evidence that active ET play an important role in regulation of follicle development, especially in the inhibition of premature luteinization of granulosa cells.
Key words: endothelin/follicular fluid/oestradiol/progesterone/testosterone
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Endothelins (ET), a family of vasoconstrictive peptides isolated originally from vascular endothelium, were demonstrated to occur widely within endocrine organs, including gonads (Yanagisawa et al., 1988
). In humans, active ET are present in three isoforms: ET-1, ET-2 and ET-3. Two and six amino acid residues of ET-1 are replaced in ET-2 and ET-3 respectively (Inoue et al., 1989). Special attention has been focused on the role of ET-1 in regulation of reproductive functions in both females and males. The action of ET-1 in the female reproductive system includes maintaining the tonus of uterine and tubal musculature, regulation of uterine blood flow as well as participation in the mechanism of progesterone withdrawal-induced vasoconstriction of spiral arteries and subsequent menstruation (Cameron and Davenport, 1992). In the hypothalamic pituitary system ET-1 and ET-3 are involved in local mechanisms controlling the gonadotrophins and prolactin secretion (Kanyicska et al., 1991). The presence of large amounts of ET-1 and ET-2 and their precursors in follicular fluid suggests a possible role in the intra-ovarian modulation of steroidogenesis and/or gamete maturation (Kamada et al., 1993; Schiff et al., 1993). Several in-vitro studies have shown that ET may inhibit the synthesis of progesterone and prevent the premature luteinization of granulosa (Tedeschi et al., 1992; Apa et al., 1998).
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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Patients
This study compromised 47 FF samples of 12 women participating in the IVF programme (mean of age 33.2 years; range 25–41). In all patients infertility was due to the tubal factor. All women were
41 years old and had a history of normal ovulatory cycles as assessed by hormonal and ultrasound criteria. In each case, hyperprolactinaemia and thyroid dysfunction were excluded. The clinical and hormonal data from the stimulated cycle are summarized in Table I. Stimulation of multiple follicles was initiated on the third cycle day by administration of human menopausal gonadotrophin (HMG; Humegon, Organon, Oss, The Netherlands) 150–450 IU i.m. daily. From the eighth day onwards follicular growth was assessed by daily measurement of plasma oestradiol and progesterone and by vaginal ultrasonography using a 5.0 MHz transducer (Bruel & Kierl, Maerum, Denmark). Human chorionic gonadotrophin (HCG; Pregnyl, Organon) 10 000 IU i.m. was given to the patients to induce ovulation when the mean diameter of the largest follicle reached 15–17 mm. Oocyte retrieval was performed 35 h after HCG injection by means of ultrasonically guided transvaginal puncture. FF from each follicle was collected separately and the longest diameter of follicle was measured on ultrasound image. The study was performed in accordance with protocol accepted by the local ethical commission.
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Only FF specimens without visible blood were used for further analysis. After oocyte isolation aprotinin (Jelfa SA, Jelenia, Gora, Poland) 500 IU/ml was added to the FF and samples were centrifuged at 1400 g for 10 min to remove cellular fragments. The samples were kept at –20°C until assayed.
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 Kolmogorow–Smirnov 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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The concentrations of 21 residue ET were evaluated in 47 samples of FF aspirated form follicles ranged from 11 to 30 mm in diameter. The analysis of the pooled samples of FF revealed marked negative correlation between active ET and follicle size (r = –0.31; P < 0.05; Figure 1A
). The significant negative correlation was observed also between ET and follicular progesterone concentrations (r = –0.56; P < 0.001; Figure 1B). A slight positive relationship was found between ET and testosterone concentrations, but the result was not significant (r = 0.25; P = 0.08). No correlation between ET and oestradiol was found in pooled samples.
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The data from individual patients were also analysed, from whom three or more samples of FF were obtained. As shown in Table II
, significant inverse correlation between ET concentration and follicle diameter was observed in all women, and correlation coefficients were higher than in pooled samples. Similarly, a strong negative relationship between ET and progesterone was observed in five of seven women, and the same number of patients ET showed significant positive correlation with testosterone. Correlation between ET and oestradiol concentrations varied among individual subjects and failed to show any general trend.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present study, using specific radioimmunoassay for 21 amino acid ET, it was possible to evaluate the concentrations of active ET in FF in relation to ovarian physiology. The primary antibody used in this method fully crossreacted with all three isoforms of ET, and therefore values presented in the present report refer to the total amount of active ET, but not to their weakly active precursors. ETA receptors are predominant in the human ovary; however, most of them are localized in the theca interna and on intra-ovarian blood vessels (Mancina et al., 1997
), and are thought to participate in mitogenic and vasoregulatory effects of ET (Kamada et al., 1995). Human luteinized granulosa cells express specific transcripts for ETA and ETB receptors and both subtypes are involved in regulation of ovarian steroidogenesis (Kamada et al., 1995; Apa et al., 1998). Because ET-1 and ET-2 shows similar affinity to both receptor subtypes (Masaki et al., 1994), all active isoforms of ET that potentially can influence ovarian steroidogenesis in vivo, were measurable with the assay used in this study.
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 5–6 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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1 To whom correspondence should be addressed
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References |
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Submitted on February 9, 1999; accepted on June 16, 1999.
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