1 Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena Policlinico Le Scotte, Viale Bracci, 53100 Siena, 2 Department of Pharmaceutical Sciences and 3 Department of Applied Mathematics, University of Padova, Siena and Padova, Italy
4 To whom correspondence should be addressed. Email: carretti{at}unisi.it
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Abstract |
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Key words: central nervous system/menstrual cycle/neuroendocrine/ovary/serotonin
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Introduction |
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In humans the precise delineation of the relationship between gonadotrophins and 5-HT function is not fully ascertained to date, and surprisingly little attention has been paid to the relationships between levels of CNS serotonin precursor concentration, as indicators of 5-HT function and activity, and modifications of LH and FSH in women (Berendsen, 2000), as well as on the timing (days, minutes?) of these putative modifications during the menstrual cycle. Indeed, to date we only know that fertile women have decreased whole brain 5-HT synthesis (Nishizawa et al., 1997
) and increased 5-hydroxyindoleacetic acid cerebrospinal fluid levels (Agren et al., 1986
).
A good tool to study the behaviour of serotonin in CNS could be represented by CNS 5-HT precursors, mainly the tryptophan levels in the serum, since it was demonstrated that in the animal model free tryptophan in serum reflects brain tryptophan level and 5-HT synthesis (Tagliamonte et al., 1973). Indeed, serum tryptophan can be measured in two forms, total (TT) and free (FT), although only FT has the property of crossing the haemato-encephalic barrier and then being converted into 5-HT in the brain. Therefore, the dynamics of FT and TT changes in the serum might represent the mirror of 5-HT utilization in CNS (Biegon et al., 1980
; Biegon and McEwen, 1982
), even if this hypothesis has not been confirmed in humans.
In the present study we aimed to evaluate whether serum levels of serotonin precursorstotal (TT) and free (FT) tryptophanchange throughout the menstrual cycle and if they correlate with estradiol (E2), LH and FSH, in order to evaluate whether a relationship exists between the peripheral levels of precursors of serotoninergic activity in CNS and the pituitaryovary axis function.
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Materials and methods |
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In all patients serum concentrations of LH, FSH, E2, TT and FT were consecutively determined during the follicular (cycle days 15, 711), mid-cycle (cycle days 1416) and luteal (cycle days 1719, 2224) phases of a spontaneous menstrual cycle. Blood samples were withdrawn through an indwelling catheter placed in the antecubital vein, after an overnight fast, at the same time in the morning (08:0009:00), then samples were centrifuged and the serum was collected and analysed. Plasma was collected and stored at 20 °C until the assay.
LH and FSH were measured in the plasma using commercially available immunoradiometric kits (Technogenetics, Italy), as well as E2 (Pantex, USA). The intra-assay coefficients of variation (CV) of LH and FSH were 4.2 and 6.9% respectively, and the inter-assay CV were 9.9 and 10.8% respectively. The intra- and inter-assay CV for E2 were 6.2 and 4.9% respectively.
Determination of total and free tryptophan in serum
The blood collected in a non-heparinized tube was allowed to clot at room temperature for 30 min and then centrifuged at 3000 g for 10 min. A 0.5 ml aliquot of the serum was kept for the determination of total tryptophan (protein bound + free) while 4 ml were introduced into Amicon model 12 ultrafiltration cell with XM-50 Diaflo membrane for determination of FT (De Antoni et al., 1980). Serum ultrafiltration was carried out at 4 °C in a controlled atmosphere of 5% CO2 and 95% N2 to maintain normal physiological pH, since the binding of tryptophan to plasma proteins is affected by pH (Eccleston, 1973
; McMenamy and Oncley, 1985
) and by freezing (Eccleston, 1973
). For the determination of FT, 0.5 ml of ultrafiltrate were collected. Tryptophan was measured according to the method of Costa et al. (1987)
by high pressure liquid chromatography.
Statistical analysis
Calculations, including statistical standardization of data, correlations, cluster analysis and graphs through spline interpolation curves were carried out using Systat Intelligent software (Wilkinson, Hill and Vang) for Macintosh, version 5.2. The classification of the five elements (cluster analysis) was made for the mean values by using both hierarchical [those of Ward and centroids, single, complete, and average weighed links) (Anderberg, 1973)] and non-hierarchical [K-averages, exchanges (Anderberg, 1973
; Hartigan, 1975
)] methods. Since all methods were concordant, for brevity we reported only data obtained with the single linkage method. P<0.05 was considered statistically significant.
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Results |
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When analysis of correlation was performed using the matrix of mean values of LH, FSH, E2, TT and FT obtained in follicular, mid-cycle and luteal phase, LH values were highly and negatively correlated to TT (r=0.636) and FT (r=0.574), as well as FSH (TT, r=0.655; FT, r=0.663); and TT and FT were strongly and positively correlated to each other (r=0.801), as were LH and FSH (r=0.935) (Table II).
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Discussion |
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However, conflicting data have been reported on the relations between 5-HT precursor and plasma LH/FSH levels. Indeed, in ewes, tryptophan infusion failed to influence ovulation rates or plasma LH/FSH concentrations (Downing et al., 1997), whilst L-5-hydroxytryptophan administration amplified pulsatile secretion of LH in fertile women (Lado-Abeal et al., 1997
) and LH and FSH plasma levels in pre-pubertal female rats (Scacchi et al., 1998
). Furthermore, data from animal models have shown that 5-HT stimulates gonadotrophin secretion in goldfish and Atlantic croaker, the two teleosts in which effects of 5-HT have been investigated in any detail, by acting on the GnRH system at the pituitary gland (Somoza and Peter, 1991
; Yu et al., 1991
; Khan and Thomas, 1992
, 1993
). In mammals, both stimulatory and inhibitory influences of 5-HT on GnRH and LH secretion have been reported, depending on the developmental stages of the animals and the experimental approach (Vitale et al., 1986
; Arias et al., 1990
; Li and Pelletier, 1995
; Hery et al., 1997
; Lado-Abeal et al., 1997
; Fink et al., 1999
). Furthermore, other studies have shown that 5-HT and its precursor 5-hydroxytryptophan (5-HTP) regulate LH secretion in mammals, including humans, by acting centrally via the stimulation of GnRH neurons (Vitale et al., 1986
; Lado-Abeal et al., 1997
; Hery et al., 1997
; Fink et al., 1999
). Selective degeneration of serotonergic nerve terminals in the ventromedial region of the hypothalamus by 5,7-dihydroxytryptamine, a 5-HT neurotoxin, results in reduced LH levels in rats (Van de Kaar et al., 1980
). In any case, the interrelations between the serotoninergic system with other pathways, such as in the case of the excitatory amino acid system, must be taken into account to explain the complex control of gonadotrophin secretion by 5-HT (Scacchi et al., 1998
).
The second result of the present study confirms that, during the first (follicular) phase of the menstrual cycle, more FT is formed from TT than during the second (luteal) phase, suggesting the availability of higher amounts of FT: in this first phase of the cycle, an acceleration would take place in the process of conversion of FT from TT in plasma and, consequently, of the FT transport from bloodstream to CNS through the haemato-encephalic barrier. In the follicular phase there is the highest negative correlation between LH and FT, and between LH and TT, much stronger than the correlation existing between the two gonadotrophins, suggesting a kind of serotoninergic blocking action on gonadotrophin release, or a common but opposite dependence on estrogen-mediated feedback mechanism. Furthermore, serum TT and FT are strictly correlated to each other, just like FSH and LH, supporting the notion of a close relationship between these two tryptophan components and that their metabolism and utilization may also be hormonally regulated during the fertile age. There are in fact at least two main steps for the conversion of plasma tryptophan to brain serotonin utilization: (i) the detachment of FT from TT in the plasma; (ii) hydroxylation of FT to 5-hydroxytryptamine (5-OHTP) and its decarboxylation to 5-HT into the brain. We might suppose: (i) a central estrogen-mediated feedback mechanism which has influence on the cyclic behaviour of plasma tryptophan consumption through the menstrual cycle and which can influence the detachment of FT from TT in the different phases (this mechanism might be thought as analogous and inverse to that of gonadotrophins); (ii) a brain mechanism of serotonin production and utilization through the control and regulation of tryptophan hydroxylase activity, since this estrogen regulation on tryptophan hydroxylase protein has been demonstrated in the dorsal raphe of macaques (Bethea et al., 2000), together with the effect of estrogens at the level of mRNA expression (Pecins-Thompson and Bethea et al., 1999
). Very recently, in the same animal region it has been seen that estrogens may increase serotonin production and transport, suggesting a complex combination of gene transcription, post-transcriptional processing and substrate feedback mechanism at the same brain level (Smith et al., 2004
). This complex body of data suggests that during the fertile age in women, estrogens, gonadotrophin release and serotonin precursors are strictly and cyclically dependent on each other. Estrogen may play a role in the preparation of CNS areas to 5-HT action as they do for gonadotrophins. Indeed, the influence of estrogens on the 5-HT pathway has been suggested by several pieces of evidence. Differences in estrogen concentration between the sexes may modulate the increased 5-HT activity seen in female rats, including higher 5-HT levels in the brain stem and limbic forebrain (Fink et al., 1996
), 5-HT synthesis (Haleem et al., 1990
), and 5-HT turnover (Rosencrans, 1970
). In addition, the effects of the estrous cycle on 5-HT function include an increase in brain 5-HT receptor concentration (Biegon et al., 1980
; Vitale et al., 1984
), 5-HT concentration (Fludder and Tonge, 1975
), and 5-HT binding (Uphouse et al., 1986
) during pro-estrous, the phase when an estrogen surge is observed. Finally, ovariectomy causes decreases in 5-HT1 binding (Biegon and McEwen, 1982
), 5-HT2A binding and expression (Zanisi and Messi, 1991
; Sumner and Fink, 1997
), and 5-HT transporter binding sites and expression (McEwen et al., 1997
), while estrogen replacement to ovariectomized rats reverses these findings. In the light of this evidence, a stimulatory role of estrogens on 5-HT synthesis in animals has been proposed.
In conclusion, data from the present study suggest that, in the serum of fertile women, the levels of TT and FT change according to the menstrual cycle and are negatively correlated to gonadotrophins, being depleted when gonadotrophin levels are elevated. Taken together, the present data and the finding that the main utilization of serotonin precursors occurs in the median raphe region, where the regulatory mechanisms of gonadal status cyclic modification is located (Klink et al., 2002), lead us to suggest that serum TT and FT are involved in FSH/LH release and that their metabolism and dynamics might be regulated by estrogens. Our study may represent an in vivo human model to investigate dynamically the functions of brain serotonin precursors, especially in relation to gonadotrophin hormones during physiological and pathological events or during SSRI drug intake.
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Submitted on July 29, 2004; resubmitted on December 14, 2004; accepted on January 15, 2005.
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