1 Department of Pediatrics/Division of Endocrinology and 2 Reproductive Sciences Program, University of Michigan,Ann Arbor, Michigan, USA, 3 Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia and 4 Oxford Brookes University, Oxford, UK
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Abstract |
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Key words: activin A/follistatin/girls/inhibin A and B/puberty
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Introduction |
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The recent advent of specific, two-site immunoassays for inhibin A and B and activin A has made it possible to measure these regulatory peptides in the peripheral circulation. Initial studies utilizing these assays have suggested an endocrine role for inhibin A and B in the control of FSH secretion during the menstrual cycle in combination with oestradiol (Groome et al., 1994, 1996
; Welt et al., 1997
; Hayes et al., 1998
). Perimenopausal women have an age-related increase in serum FSH concentration that precedes a decline in serum oestradiol concentration (Yen, 1991
). In these women, activin A concentrations increase while inhibin B concentrations decrease, suggesting that the sum change in concentrations of positive regulators of FSH (activin and GnRH), and negative regulators of FSH (oestradiol, inhibin and follistatin) may together contribute to the increases in FSH secretion seen in ageing (Reame et al., 1998
).
Whether gonadal peptides regulate FSH secretion during pubertal maturation is unclear. Recent studies have shown that inhibin B concentrations are greater in prepubertal boys than in prepubertal girls, suggesting that the greater FSH concentrations seen in prepubertal girls could be related to less inhibin availability from the developing ovary (Hayes et al., 1998). Prepubertal girls, unlike prepubertal boys, readily release FSH into the circulation in response to GnRH administration (Lee, 1985
; Foster et al., 1992
). Ovarian size increases throughout the prepubertal period and into puberty as a consequence of increased stroma, growth of individual follicles and growth of number of follicles (Peters et al., 1978
). Thus, the developing gonad could produce increasing concentrations of gonadal peptides that in turn could affect FSH secretion. Since activin and follistatin are produced in the pituitary (Ying, 1988
; DePaolo et al., 1991
), there is also the possibility that changes in the peripheral concentrations of activin and follistatin may reflect the development of an intra-pituitary feedback system that regulates FSH secretion (Bilezikjian et al., 1994
). This study was designed to determine whether the gonadal peptides, activin A, inhibin A, inhibin B and follistatin, are correlated with maturation markers in girls. Such a correlation would suggest that gonadal peptides could contribute to the changes in FSH concentration that occur at puberty.
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Materials and methods |
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Hormone assays
Activin A and inhibins A and B were determined using two-site enzyme-linked immunosorbent assays (ELISA) (Serotec, Raleigh, NC, USA). The sensitivities of the assays were 0.2 ng/ml, 5 pg/ml and 16 pg/ml for activin A, inhibin A and inhibin B respectively. The samples were measured in one assay in triplicate. The intra-assay coefficients of variation were 5, 8 and 6% for activin A, inhibin A and inhibin B respectively.
Total follistatin concentrations were determined using a heterologous radioimmunoassay described previously (O'Connor et al., 1999) which employs dissociating reagents [20% Tween 20, 10% sodium deoxycholate and 0.4% sodium dodecyl sulphate (SDS)], to remove the interference of bound activin. The rabbit polyclonal antiserum used in this assay was raised against 35 kDa bovine follistatin. In the assay, recombinant human follistatin (rhFS) 288 was used as both tracer and standard. Cross-reactivity was 100% for rhFS 288 and 36% for rhFS 315 (O'Connor et al., 1999
). In the presence of dissociating reagents, addition of increasing amounts of activin did not alter the amount of measured follistatin (O'Connor et al., 1999
). The assay sensitivity was 1.6 ng/ml, and the intra-assay coefficient of variation 12.3%. Samples of human male and female serum diluted parallel in the assay, and the recovery of added follistatin (3.125100 ng/ml) ranged from 103 to 110% (O'Connor et al., 1999
). Follistatin 288 concentrations were measured using a recently developed two-site ELISA (Evans et al., 1998
). The assay sensitivity was 37 pg/ml and the intra-assay coefficient of variation 17%. This assay cross-reacts 9.9% with follistatin 315 (Evans et al., 1998
). Free follistatin was determined by a second-generation two-site chemiluminescent assay that utilizes two monoclonal antibodies generated against non-overlapping epitopes of human follistatin as described previously (McConnell et al., 1998
). The assay sensitivity was 0.8 ng/ml, and the intra-assay coefficient of variation <4%.
LH and FSH were determined by immunofluorometric assay using commercially available kits (Wallac, Gaithersburg, MD, USA). The assay sensitivity was 0.05 IU/l for LH and FSH. The intra-assay coefficients of variation were 3.1% for LH and 3.9% for FSH.
Statistical analyses
Hormone concentrations were transformed logarithmically before analysis to adjust for data heterogeneity. Differences in hormone concentrations between pubertal stages were assessed by one-way analysis of variance followed by a Duncan new multiple range test. Correlations were determined by regression. Adult volunteers did not have bone age determinations; their chronological age was assigned to the bone age value. In all cases, a P value of < 0.05 was considered significant.
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Results |
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Follistatin concentrations
Total follistatin concentrations had an apparent decrease between pubertal stages I to IV and stage V puberty (Figure 1), exhibiting a significant decrease with advancing bone age (r = 0.634; P = 0.0001; Table II
). Free follistatin concentrations were near the limit of assay detection in all subjects (Figure 1
). When determined by an ELISA specific for follistatin 288, follistatin concentrations exhibited a peak in stage III puberty and declined significantly thereafter. Analysis of the relationship between follistatin 288 concentration and bone age showed that r = 0.321 (not significant).
Correlation of gonadal peptides with FSH and oestradiol
FSH concentrations demonstrated a positive correlation with increasing concentrations of oestradiol and LH (Figure 1, Table II
). There was a positive correlation between inhibin B concentration and FSH concentration, but no significant correlations between inhibin A, activin A, or follistatin and FSH concentrations (Table II
). There were positive correlations between oestradiol concentration and inhibin A and B concentrations, but not with activin A concentration (Table II
). Total follistatin concentration had a significant negative correlation with oestradiol concentration, but there was no correlation between oestradiol concentration and follistatin 288 concentration (Table II
).
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Discussion |
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Changes in inhibins in this study agree in general with those observed in earlier studies. In both boys and girls, inhibin concentrations are lowest during pubertal stage I (Andersson et al., 1997). However, the mid-pubertal peak in inhibin B that was noted in the girls in the current study has not been observed in boys or in a previously reported longitudinal study in girls (Crofton et al., 1997
). It is difficult to make any meaningful comparisons between the current and the published report (Crofton et al., 1997
), as there were four girls in stage III puberty available for inclusion in the current study, while only one girl had a sample obtained at stage III puberty in the study of Crofton et al. Further, earlier studies (Andersson et al., 1997
; Crofton et al., 1997
) utilized single samples in their design, while the current study design incorporated an integrated measure over a 10 h period at night when gonadotrophin secretion is most active in pubertal children (Penny et al., 1977
; Foster et al., 1989
; Cemeroglu et al., 1996
; Kletter et al., 1997
). It is also of interest that in an earlier study (Boepple et al., 1996
), inhibin B concentrations found in girls with precocious puberty were similar to those in adult women, and inhibin B concentration declined when gonadotrophin secretion was down-regulated with GnRH agonist therapy. Since inhibin B is found in granulosa cells of developing follicles (Roberts et al., 1993
), the general increase in numbers of developing follicles during mid puberty may contribute to the increased concentrations of inhibin B observed in the mid-pubertal girls of the current investigation. Studies relating inhibin B concentrations to changes in follicular size and oestradiol and progesterone concentrations in the pubertal transition may shed additional light on how gonadal peptide concentrations change with ovarian development.
As both inhibin A and B suppress FSH secretion (Ying, 1987), the sum total of inhibin A and inhibin B should provide an index of total peripheral inhibin input. Estimation of total inhibin by summing inhibin A and inhibin B concentrations suggests that total inhibin concentrations increase through mid puberty and decline thereafter. The directionality of inhibin changes is dictated by the higher circulating concentrations of inhibin B. Until it is established that these two immunoassays predict the protein mass of inhibin A and B correctly, and it is demonstrated that the relative biological potency of these two isomers in suppressing human FSH secretion is similar, this estimate should be viewed with caution.
Correlation data between hormone concentrations do not imply a cause-and-effect relationship, but are of interest in determining whether a relationship may be present. Hence, the correlation coefficients between the gonadal peptides and FSH and oestradiol concentrations were determined in the subject population of this study. Previously, it was reported that both inhibin A and B concentrations exhibit positive correlations with oestradiol and FSH concentrations (Crofton et al., 1997). In the current study, only inhibin B and not inhibin A concentrations exhibited a significant correlation with FSH. The positive correlation between inhibin B and FSH suggests that the increase in FSH may actually drive the increase in inhibin B andat least during pubertyinhibin B may not be negatively regulating FSH secretion. Such a relationship is also seen with oestradiol and gonadotrophin secretion.
FSH is believed to increase oestradiol secretion during puberty, and the profound negative feedback of oestradiol on gonadotrophin secretion seen in prepubertal children is lost during puberty. This appears to be due to a change in neurotransmitter control of GnRH secretion which results in resistance of the hypothalamus to negative feedback. As puberty progresses, the pituitary gonadotroph assumes increasing importance over the hypothalamus as the site of sex steroid negative feedback (Kletter et al., 1992; Cemeroglu et al., 1998
). This change of feedback appears to permit the increase of gonadotrophin secretion seen at puberty. If gonadal peptides exhibit their primary endocrine effects at the level of the pituitary, negative feedback may not be possible until gonadotrophin secretion from the pituitary is well established, as in late puberty. As inhibin A and B concentrations both increase significantly with the increase in oestradiol concentrations, and inhibin B concentrations increase significantly with FSH concentrations, these data suggest that inhibin concentration may serve as a developmental marker of ovarian follicle development or increase in follicle number during puberty.
The cross-sectional data in this study suggest that activin concentrations do not change significantly with advancing puberty. Activin assays are relatively new (Knight et al., 1996), and hence this is the first report on changes in activin concentrations during the onset of puberty. Surprisingly, total follistatin concentrations declined steadily with advancing bone age, and follistatin 288 concentrations (determined by a separate ELISA) were also at their lowest values in late pubertal girls and adult women. Follistatin has not been shown to vary during pubertal maturation in other studies (Kettel et al., 1996
), but this may be due to differences in assays for this peptide and how the assay recognizes the different variants of follistatin. Free follistatin concentrations were at or near the assay sensitivity in all of the subjects in this study, indicating that virtually all of the circulating follistatin is activin-bound. These results also corroborate the earlier finding that almost all of the free follistatin appears to be activin-bound in the circulation of adult cycling women (McConnell et al., 1998
). As activin A concentrations appear to be constant, the decline in follistatin concentrations during late puberty suggests that more activin A may be available to regulate FSH secretion in late puberty. Animal studies have shown that activin at concentrations as low as 0.01 nmol/l can stimulate FSH secretion (Bilezikjian et al., 1998
). In the absence of measures of activin B and AB, which are also produced by the ovary, the total activin drive cannot be estimated.
Since the ovary is increasing in size with advancing puberty, the finding that total follistatin concentration declined during pubertal maturation was unexpected. Follistatin 288 concentrations also exhibited a decline in late puberty, but the correlation with bone age was not as strong. A number of studies have suggested that the ovary is not a significant source of follistatin entering into the circulating pool (Klein et al., 1993; Khoury et al., 1995
). Therefore the observation that follistatin concentrations declined during pubertal maturation may reflect changes in the paracrine regulatory loop involving activin and follistatin in the pituitary (Phillips and de Kretser, 1998
). This notion is also consistent with the significant inverse correlation between total follistatin concentrations and oestradiol concentration in the present study, and the observation that the pituitary is a net secretor of follistatin into the circulation (Phillips and de Kretser, 1998
). The fact that total follistatin concentration has a significant inverse correlation with oestradiol concentration suggests that increasing oestradiol concentrations with advancing puberty might be responsible for suppressing follistatin, but additional studies will be needed to demonstrate a cause-and-effect relationship.
The data in this study, coupled with other recent findings, indicate that inhibin concentrations vary dynamically with pubertal maturation. Changes in inhibins during pubertal maturation seem to be a reflection of ovarian maturation rather than an indicator of FSH feedback regulation. Activin A concentrations do not vary significantly with pubertal maturation, but the decline in follistatin concentrations with increasing pubertal maturation suggests that more activin could be available to increase pituitary FSH release as the ovary develops. Analysis of dynamic changes of all gonadal peptides will be needed to understand whether and how these peptides function in the complex regulation of gonadotrophin secretion.
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Acknowledgments |
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Notes |
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* Presented at the 80th meeting of The Endocrine Society, New Orleans, Los Angeles, June 1998.
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References |
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Bilezikjian, L.M., Corrigan, A.Z. and Vale, W.W. (1994) Activin-B, inhibin-B and follistatin as autocrine/paracrine factors of the rat anterior pituitary. In Burger, H.G., Findlay, J., Robertson, D. et al. (eds), Inhibin and Inhibin-related Peptides. Ares-Serono Symposia Publications, Rome, pp. 8199.
Bilezikjian, L.M., Turnbull, A.V., Corrigan, A.Z. et al. (1998) Interleukin-1beta regulates pituitary follistatin and inhibin/activin betaB mRNA levels and attenuates FSH secretion in response to activin-A. Endocrinology, 139, 33613364.
Boepple, P.A., Sluss, P.M., Khoury, R.H. and Crowley, W.F., Jr (1996) Follistatin (FS), and inhibin A and B in central precocious puberty (CPP): impact of GnRH agonist (GnRHa)-induced pituitary desensitization [Abstract 491]. Pediatr. Res., 39, 84A.
Cataldo, N.A., Rabinovici, J., Fujimoto, V.Y. and Jaffe, R.B. (1994) Follistatin antagonizes the effects of activin-A on steroidogenesis in human luteinizing granulosa cells. J. Clin. Endocrinol. Metab., 79, 272277.[Abstract]
Cemeroglu, A.P., Foster, C.M., Warner, R. et al. (1996) Comparison of the neuroendocrine control of pubertal maturation in girls and boys with spontaneous puberty and in hypogonadal girls. J. Clin. Endocrinol. Metab., 81, 43524357.[Abstract]
Cemeroglu, A.P., Kletter, G.B., Brown, M.B. et al. (1998) In pubertal girls, naloxone fails to reverse the suppression of luteinizing hormone secretion by estradiol. J. Clin. Endocrinol. Metab., 83, 35013506.
Crofton, P.M., Illingworth, P.J., Groome, N.P. et al. (1997) Changes in dimeric inhibin A and B during normal early puberty in boys and girls. Clin. Endocrinol., 46, 109114.[ISI][Medline]
DePaolo, L.V., Bicsak, T.A., Erickson, G.F. et al. (1991) Follistatin and activin: a potential intrinsic regulatory system within diverse tissues. Proc. Soc. Exp. Biol. Med., 198, 500512.[Medline]
de Winter, J.P., ten Dijke, P., de Vries, C.J. et al. (1996) Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors. Mol. Cell. Endocrinol., 116, 105114.[ISI][Medline]
Draper, L.B., Matzuk, M.M., Roberts, V.J. et al. (1998) Identification of an inhibin receptor in gonadal tumors from inhibin -subunit knockout mice. J. Biol. Chem., 273, 398403.
Evans, L.W., Muttukrishna, S. and Groome, N.P. (1998) Development, validation and application of an ultra-sensitive two-site enzyme assay for human follistatin. J. Endocrinol., 156, 275282.
Foster, C.M., Hassing, J.M., Mendes, T.M. et al. (1989) Testosterone infusion reduces nocturnal LH pulse frequency in pubertal boys. J. Clin. Endocrinol. Metab., 69, 12131220.[Abstract]
Foster, C.M., Hopwood, N.J., Beitins, I.Z. et al. (1992) Evaluation of gonadotropin responses to synthetic gonadotropin-releasing hormone in girls with idiopathic hypopituitarism. J. Pediatr., 121, 528532.[ISI][Medline]
Greulich, W.W. and Pyle, S.I. (1955) Atlas of Skeletal Development of the Hand and Wrist. Stanford University Press, Stanford.
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1994) Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. Clin. Endocrinol. (Oxf.), 40, 717723.[ISI][Medline]
Groome, N.P., Illingworth, P.J., O'Brien, M. et al. (1996) Measurement of dimeric inhibin B throughout the human menstrual cycle. J. Clin. Endocrinol. Metab., 81, 14011405.[Abstract]
Hayes, F.J., Hall, J.E., Boepple, P.A. and Crowley, W.F. Jr (1998) Differential control of gonadotropin secretion in the human: endocrine role of inhibin. J. Clin. Endocrinol. Metab., 83, 18351841.
Kettel, L.M., DePaolo, L.V., Morales, A.J. et al. (1996) Circulating levels of follistatin from puberty to menopause. Fertil. Steril., 65, 472476.[ISI][Medline]
Khoury, R.H., Wang, Q.F., Crowley, W.F. Jr et al. (1995) Serum follistatin levels in women: evidence against an endocrine function of ovarian follistatin. J. Clin. Endocrinol. Metab., 80, 13611368.[Abstract]
Klein, R., Findlay, J.K., Clarke, I.J. et al. (1993) Radioimmunoassay of FSH-suppressing protein in the ewe: concentrations during the oestrous cycle and following ovariectomy. J. Endocrinol., 137, 433443.[Abstract]
Kletter, G.B., Foster, C.M., Beitins, I.Z. et al. (1992) Acute effects of testosterone infusion and naloxone on luteinizing hormone secretion in normal men. J. Clin. Endocrinol. Metab., 75, 12151219.[Abstract]
Kletter, G.B., Padmanabhan, V., Beitins, I.Z. et al. (1997) Effects of estradiol infusion and naloxone on LH secretion in pubertal boys. J. Clin. Endocrinol. Metab., 82, 40104014.
Knight, P.G., Muttukrishna, S. and Groome, N.P. (1996) Development and application of a two-site immunoassay for the determination of `total' activin-A concentrations in serum and follicular fluid. J. Endocrinol., 148, 267279.[Abstract]
Lee, P.A. (1985) Neuroendocrine maturation and puberty. In Lavery, J.P. and Sanfilippo, J.S. (eds), Pediatric and Adolescent Obstetrics and Gynecology. Springer-Verlag, New York, pp. 1226.
McConnell, D.S., Wang, Q.F., Sluss, P.M. et al. (1998) A two-site chemiluminescent assay for activin-free follistatin reveals that most follistatin circulating in men and normal cycling women is in an activin-bound state. J. Clin. Endocrinol. Metab., 83, 851858.
O'Connor, A.E., McFarlane, J.R., Hayward, S. et al. (1999) Serum activin A and follistatin levels during human pregnancy: a cross-sectional and longitudinal study. Hum. Reprod., 14, 827832.
Penny, R., Olambiwonnu, N.O. and Frasier, S.D. (1977) Episodic fluctuations of plasma gonadotropins in pre- and post-pubertal girls and boys. J. Clin. Endocrinol. Metab., 45, 307311.[Abstract]
Peters, H., Byskov, A.G. and Grinsted, J. (1978) Follicular growth in fetal and prepubertal ovaries in humans and other primates. Clin. Endocrinol. Metab., 7, 469485.[ISI][Medline]
Phillips, D.J. and de Kretser, D.M. (1998) Follistatin: a multifunctional regulatory protein. Front. Neuroendocrinol., 19, 287322.[ISI][Medline]
Reame, N.E., Wyman, T.L., Phillips, D.J. et al. (1998) Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cycling women. J. Clin. Endocrinol. Metab., 83, 33023307.
Roberts, V.J., Barth, S., Elroely, A. and Yen, S.S.C. (1993) Expression of inhibin/activin subunits and follistatin messenger ribonucleic acids and proteins in ovarian follicles and the corpus luteum during the human menstrual cycle. J. Clin. Endocrinol. Metab., 77, 14021410.[Abstract]
Tanner, J.M. (1978) Growth at Adolescence. Blackwell, Oxford.
Welt, C.K, Martin, K.M., Taylor, A.E. et al. (1997) Frequency modulation of follicle-stimulating hormone (FSH) during the luteal-follicular transition: evidence for FSH control of inhibin B in normal women. J. Clin. Endocrinol. Metab., 82, 26452652.
Xu, J., McKeehan, K., Matsuzaki, K. and McKeehan, W.L. (1995) Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism. J. Biol. Chem., 270, 63086313.
Yen, S.S.C. (1991) The human menstrual cycle: neuroendocrine regulation. In Yen, S.S.C. and Jaffe, R.B. (eds), Reproductive Endocrinology. Saunders, Philadelphia, pp. 273308.
Ying, S.-Y. (1987) Inhibins and activins: chemical properties and biological activity. Proc. Soc. Exp. Biol. Med., 186, 253264.[Abstract]
Ying, S.-Y. (1988) Inhibins, activins and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. Endocr. Rev., 9, 267293.[Abstract]
Submitted on July 19, 1999; accepted on January 27, 2000.