Sex differences in FSH-regulatory peptides in pubertal age boys and girls and effects of sex steroid treatment

Carol M. Foster1,3, Pamela R. Olton1, Michael S. Racine1, David J. Phillips2 and Vasantha Padmanabhan1

1 Department of Pediatrics/Endocrinology and the Reproductive Sciences Program, University of Michigan, Ann Arbor, MI 48109, USA and 2 Institute of Reproduction and Development, Monash University, Clayton, Victoria, 3168 Australia

3 To whom correspondence should be addressed at: D1205 MPB Box 0718, Ann Arbor, MI 48109, USA. e-mail: cmfoster{at}umich.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: FSH concentrations are higher in girls than in boys before puberty. We hypothesized that steroid-mediated changes in FSH-regulatory proteins underlie the sex differences in FSH secretion and pubertal timing. METHODS: FSH-regulatory proteins, LH, FSH and sex steroids were measured in five boys, 10 girls, and five girls with Turner syndrome before and during sex steroid treatment (girls, 0.05 mg/day estradiol; boys, 5 mg/day testosterone) for up to 4 weeks. Blood was obtained every 15 min from 20.00 to 08.00 h before and during sex steroid treatment. RESULTS: The mean FSH concentration was higher in girls than in boys (P = 0.0044). Activin-A concentrations were greater (P < 0.0001) and inhibin-B concentrations lower (P < 0.0001) in girls compared with boys. Steroid treatment (i) suppressed LH/FSH concentrations in all subjects; (ii) increased the mean activin-A concentration in all but the Turner girls (P = 0.001); and (iii) decreased inhibin-B concentrations in boys (P = 0.005) but not in girls. Total follistatin and follistatin 288 concentrations did not differ by sex. CONCLUSIONS: Sex steroids regulate circulating activin-A and inhibin-B concentrations in children. The lower inhibin-B and higher activin-A concentrations may explain the higher FSH and earlier onset of puberty in girls.

Key words: activin/follistatin/FSH/inhibin/puberty


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Girls begin puberty earlier and have as much as a 5-fold increase in incidence of precocious puberty as do boys (Grumbach and Styne, 1998Go). The reasons for the sex differences in the timing of puberty are not known. Puberty is heralded by an increase in the episodic release of LH under the control of increased GnRH pulse frequency and amplitude (Marshall and Kelch, 1986Go). It has been thought that sex differences in central nervous system restraint of GnRH and subsequently of LH secretion account for the differences in timing of onset of puberty in boys and girls. FSH secretion is readily detected prior to the onset of puberty and exhibits sexual dimorphism in basal and GnRH-stimulated concentrations (Winter, 1982Go; Lee, 1985Go). Thus, assessment of factors regulating FSH secretion during childhood may enhance our understanding of sex differences in pubertal development and the pathogenesis of precocious puberty.

Although FSH is secreted episodically under the control of GnRH, FSH is also secreted constitutively under the control of a group of peptides collectively known as the FSH-regulatory proteins (McNeilly, 1988Go; Ying, 1988Go; Muyan et al., 1994Go; Farnsworth, 1995Go; Padmanabhan and Sharma, 2001Go; Padmanabhan and West, 2001Go). These peptides include the activins and inhibins, members of the transforming growth factor-{beta} (TGF-{beta}) family, and the follistatins. The activins stimulate FSH secretion (Ying, 1988Go) and are made in the gonads, pituitary and extragonadal tissue (DePaolo et al., 1991Go; Mather et al., 1992Go). Activin-A, comprised of two {beta}A subunits, is the only activin peptide for which relevant serum measurements have been available (Knight et al., 1996Go). Although activins have been thought to have a primary paracrine/autocrine role on FSH release, serum concentrations of activin-A vary with some reproductive changes, suggesting that activin-A may have endocrine effects. Activin-A varies during ageing (Loria et al., 1998Go; Reame et al., 1998Go) and, in some studies, during the menstrual cycle (Muttukrishna et al., 1996Go). Activin-A concentrations have been reported to remain stable during pubertal maturation in boys and girls (Foster et al., 2000Go; Luisi et al., 2001Go).

Inhibins suppress FSH secretion (Ying, 1988Go; Welt et al., 2002Go). They are structurally related to the activins but contain an {alpha}-subunit in combination with an activin {beta}-subunit (Ying, 1988Go). Inhibin-A and inhibin-B are both produced in the ovaries, while the testis produces only inhibin-B (Roberts et al., 1993Go; Anawalt et al., 1996Go; Hayes et al., 1998Go). Serum inhibin-A concentrations are high in the 3 months after birth in girls and then decline to low concentrations until late puberty (Bergada et al., 1999Go; Foster et al., 2000Go; Sehested et al., 2000Go). Inhibin-A is produced principally in dominant ovarian follicles (Roberts et al., 1993Go; Welt et al., 2002Go). Serum concentrations of inhibin-B are high for up to 3 months after birth in boys and girls and then decline to low concentrations in girls until the time of puberty, when concentrations then increase again (Foster et al. 2000Go; Sehested et al., 2000Go; Crofton et al., 2002aGo). Inhibin-B is associated with small antral follicles (Roberts et al., 1993Go; Welt et al., 2002Go). Boys have much higher concentrations of inhibin-B than do girls throughout childhood and exhibit a further increase in inhibin-B concentrations with the onset of puberty (Andersson et al., 1997Go; Crofton et al., 2002bGo).

The third FSH regulator, follistatin, is a monomeric peptide that binds activin and prevents activin binding to its receptor, thereby inhibiting FSH secretion (Robertson, 1992Go; de Winter et al., 1996Go; Phillips and deKretser, 1998Go). Processing of mRNA results in at least two forms of follistatin, a 315 kDa peptide and a 288 kDa peptide (Robertson, 1992Go; Phillips and deKretser, 1998Go). Follistatins are made in the gonads, pituitary and extragonadal tissues (Robertson, 1992Go; Phillips and deKretser, 1998Go).

In this study, we hypothesized that the sex differences in FSH concentrations are due to differences in the concentrations of FSH-regulatory peptides, specifically expecting the inhibitory regulators, inhibins and/or follistatins, to be lower and/or the stimulatory regulator, activin, to be higher in girls than in boys. Previously, we had observed that, in girls, follistatin concentrations decline in late puberty in conjunction with an increase in serum estradiol concentrations (Foster et al., 2000Go). Thus, we hypothesized that sex steroids might be involved in the regulation of the serum concentrations of the FSH-regulatory peptides. The approach we took to address this premise was to raise the sex steroid concentrations of boys and girls to adult levels by treating pubertal age children with transdermal testosterone or estradiol. To determine the effects of sex steroids on FSH-regulatory peptides originating from extragonadal sites, we also examined the effect of estradiol treatment in girls with gonadal dysgenesis who might be expected to exhibit little or no gonadal peptide secretion.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
We studied 10 girls, five boys and five girls with Turner syndrome. Their clinical characteristics are shown in Table I. A sixth boy, age 13.2 years with bone age 11.5 years in stage II puberty, who did not complete the sex steroid treatment protocol, was included only in the boys’ basal hormone determinations. All children were in good health and receiving no medications. The boys and the girls without Turner syndrome all had either short stature or constitutional growth delay. None of these children were found to have growth hormone deficiency or an abnormality of gonadotrophin secretion. With the exception of the girls with Turner syndrome, all of the children have exhibited spontaneous pubertal advancement and continued normal growth.


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Table I. Clinical characteristics of subjects treated with sex steroids
 
Protocol
Studies were carried out in the General Clinical Research Center (GCRC) of the University of Michigan. The University of Michigan Institutional Review Board approved the research protocol, and informed written consent was obtained from a parent and assent from the child prior to the study.

Children underwent research studies as a part of an evaluation for short stature and/or adolescent delay. Children were admitted to the GCRC at 17.00 h. A catheter was placed in a forearm vein to serve as a venous access. Blood was obtained every 15 min beginning at 20.00 h for 12 h until 08.00 h the following day. At the completion of the first GCRC admission, girls were treated with transdermal estradiol (0.05 mg/24 h systems, Alora®, Watson Pharma, Inc., Corona, CA). New patches were applied every 4 days. All pubertal girls were treated for 1 week, except subject 9. Subject 9 and the five girls with Turner syndrome were treated for 4 weeks. Boys were discharged on 5 mg/day transdermal testosterone (Androderm®, Watson Pharma, Inc.) and treated for 4 weeks. Testosterone patches were changed daily. All children were then readmitted to the GCRC and studied with the same protocol as used before treatment. Blood withdrawal was minimized by pooling equal aliquots of serum from each sample to make four pools covering the following time blocks: 20.00–22.45 h; 23.00–01.45 h; 02.00–04.45 h; and 05.00–08.00 h. For the girls with Turner syndrome, a single overnight pool sample was assayed. Each pool sample was assayed for LH, FSH, estradiol or testosterone, activin-A, immunoreactive inhibin (Monash assay), inhibin-B, total follistatin and follistatin 288. Inhibin-A was determined in girls only.

Hormone assays
Activin-A, inhibin-A and inhibin-B were determined using two-site enzyme-linked immunosorbent assays (ELISAs) from Serotec (Raleigh, NC). The assay sensitivity for activin-A was 0.04 ng/ml, and the intra- and inter-assay coefficients of variation (CVs) were 6 and 16%, respectively. The assay sensitivity for inhibin-A was 8 pg/ml, and the intra- and inter-assay CVs were 6 and 13%. The assay sensitivity for inhibin-B was 15 pg/ml, and the intra- and inter-assay CVs were 10 and 16%. Follistatin 288 was measured using a recently developed two-site ELISA (Evans et al., 1998Go). The assay sensitivity was 37 pg/ml, and the intra- and inter-assay CVs were 8 and 16%, respectively. This assay cross-reacts 9.9% with follistatin 315. All samples for one child were analysed in the same assay.

Total immunoreactive inhibin was measured by a heterologous radioimmunoassay as described previously (Robertson et al., 1988Go) using an in-house human recombinant (hr) inhibin-A as both standard and tracer. The assay cross-reacts 288% with pro-{alpha}C, the pro-sequence of the inhibin {alpha}-subunit (Robertson et al., 1989Go). The intra- and inter-assay CVs were 7.8 and 7.9%, respectively, and the limit of detection was 0.1 ng/ml.

Total follistatin concentrations were determined using a heterologous radioimmunoassay described previously (O’Connor et al., 1999Go) which employs dissociating reagents (20% Tween-20, 10% sodium deoxycholate and 0.4% SDS), to remove the interference of bound activin. The rabbit polyclonal antiserum used in this assay was raised against 35 kDa bovine follistatin (FS). In the assay, hrFS 288 was used as both tracer and standard. Cross-reactivity is 100% for hrFS 288 and ~33% for hrFS 315. The assay sensitivity was 1.66 ng/ml, and the intra- and inter-assay CVs were 6.7 and 4.8%, respectively.

Estradiol was determined by radioimmunoassay using kits purchased from Diagnostic Products Corporation (Los Angeles, CA). The assay sensitivity was 18 pmol/l and the intra- and inter-assay CVs were 5 and 9%, respectively. Testosterone was determined by DELFIA immunofluorometric assay using kits purchased from Wallac-PerkinElmer (Gaithersburg, MD). The assay sensitivity was 0.4 nmol/l, and the intra- and inter-assay CVs were 5 and 8%, respectively.

LH and FSH were determined by immunofluorometric assay using kits purchased from Wallac-PerkinElmer. The assay sensitivity was 0.05 IU/l for LH and FSH. The intra-assay CVs were 3.1% for LH and 3.9% for FSH. The inter-assay CVs were 6.1% for LH and 4.8% for FSH.

Statistical analyses
All hormone values were transformed logarithmically prior to analysis. Comparisons of time on hormone concentration were made by repeated measures analysis of variance (ANOVA). Single comparisons before and after sex steroid therapy were made by paired Student’s t-test. A P-value of <0.05 was considered to be significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sex differences in FSH and its regulatory peptides
Mean FSH and FSH-regulatory peptide concentrations were compared between six boys and 10 girls as shown in Figure 1. As expected, the mean overnight FSH concentrations were significantly greater in the girls than in the boys (P < 0.005), while total inhibin and inhibin-B concentrations were greater in the boys than in the girls (P = 0.002 for total inhibin and P < 0.0001 for inhibin-B). Activin-A was also significantly greater in girls than in boys (P < 0.0001). Neither total follistatin nor follistatin 288 varied by sex. Regression analyses of the relationship between activin-A and log FSH concentration, combining data from boys and girls, was not significant (r2 = 0.204; P = 0.07), while the correlation between inhibin-B and log FSH concentration was highly significant (r2 = 0.463; P = 0.005).



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Figure 1. Sex differences in gonadotropin and FSH-regulatory peptide concentrations. The results shown are the means ± SE of the determinations made in four pools comprised of 3 h time blocks throughout the night. Significant differences between boys and girls are denoted by the P-value shown in the figures.

 
Estradiol treatment in pubertal girls
The mean changes in the factors assayed between 20.00 and 08.00 h before and during estradiol treatment in the 10 girls are shown in Figure 2. Prior to estradiol treatment, mean LH concentration increased significantly from 20.00–23.00 h to 23.00–02.00 h, and remained increased at 02.00–05.00 h (P < 0.001). FSH concentration also increased significantly (P < 0.005) from 20.00 to 23.00 h and 23.00 to 02.00 h, and remained increased in the remaining time blocks. Activin-A concentrations declined significantly (P = 0.024) from 20.00–23.00 h to 05.00–08.00 h. Total inhibin, inhibin-B, total follistatin and follistatin 288 concentrations remained unchanged overnight with respect to clock time.



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Figure 2. Estradiol, gonadotropin and FSH-regulatory peptides in girls before and during estradiol treatment. The results shown are the means ± SE of the determinations made in four pools comprised of 3 h time blocks throughout the night. Significant differences (P < 0.05) between control and treatment are shown by brackets with asterisks. Differences by time block are shown by letter: a = a difference from the 20.00 to 22.45 h time block; b = a difference from the 23.00 to 01.45 h time block.

 
Estradiol treatment increased the mean overnight estradiol concentrations from 20 ± 2 to 101 ± 36 pmol/l (P = 0.015), and suppressed the night time increase in LH concentration, decreasing the mean overnight LH concentration from 1.18 ± 0.56 to 0.06 ± 0.01 IU/l (P < 0.0001). Estradiol treatment suppressed FSH in all time blocks to 0.2 IU/l (P < 0.0001). It is of interest that estradiol treatment increased activin-A concentrations in each time block (P < 0.0001) such that the mean overnight activin-A concentration increased from 231 ± 19 to 290 ± 31 pg/ml. However, even with estradiol treatment, activin-A concentration declined significantly from 20.00–23.00 h in each succeeding time block to 05.00–08.00 h (P = 0.0006). Neither immunoreactive inhibin, inhibin-B, total follistatin nor follistatin 288 concentrations were affected significantly by estradiol treatment. Only two girls had measurable concentrations of inhibin-A before estradiol treatment. In those two girls, the mean inhibin-A concentration decreased from 19.4 and 18.0 pg/ml before estradiol treatment to 9.8 and 7.8 pg/ml after estradiol treatment.

Estradiol treatment in girls with Turner syndrome
The mean changes in FSH, inhibin-B, activin-A and estradiol concentration before and during estradiol treatment are shown in Figure 3. Estradiol concentrations increased from 18 to 99 ± 29 pmol/l (P = 0.004) and mean FSH concentrations declined from 103 ± 11 to 12.3 ± 6 IU/l following transdermal delivery of estradiol. Activin-A concentrations were not different in these girls before and during estradiol treatment. Inhibin-A and -B concentrations were at the assay detection limit both before and during estradiol treament, and follistatin 288 was not measured because of insufficient sample volume.



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Figure 3. FSH, estradiol and activin-A concentrations in girls with Turner syndrome before and during estradiol treatment. Results are the means ± SE of determinations made in overnight pools.

 
Testosterone treatment in boys
Testosterone treatment increased the mean serum testosterone concentration as evidenced by the significant suppression of LH and FSH concentrations compared with pre-treatment concentrations (Figure 4). Activin-A concentrations increased significantly with testosterone treatment from 111 ± 10 to 164 ± 13 pg/ml (P = 0.001). Inhibin-B concentrations decreased with testosterone treatment from 200 ± 15 to 123 ± 19 pg/ml (P = 0.005), but immunoreactive inhibin concentrations were not different with testosterone treatment. Neither the mean total follistatin nor the mean follistatin 288 concentration changed with testosterone treatment.



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Figure 4. Testosterone, gonadotropin and FSH-regulatory peptides in boys before and during testosterone treatment. Results are the means ± SE of the determinations made in four pools comprised of 3 h time blocks throughout the night. Significant differences (P < 0.05) between control and treatment are shown by brackets with asterisks. Differences by time block are shown by letter: a = a difference from the 20.00 to 22.45 time block; b = a difference from the 23.00 to 01.45 time block; c = a difference from the 02.00 to 04.45 time block.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Secondary sexual characteristics develop ~18 months earlier in girls than in boys (Tanner, 1978Go), but why this occurs is unknown. Both sexes exhibit a similar increase in LH and, by inference, GnRH secretion at the onset of puberty (Hale et al., 1988Go; Cemeroglu et al., 1996Go), suggesting that the cause(s) of sex differences in pubertal timing lie elsewhere than in the regulation of GnRH secretion. Unlike LH, FSH secretion exhibits sexual dimorphism in childhood and in early puberty (Winter, 1982Go; Lee, 1985Go). FSH is important in the growth of gonads in both boys and girls and plays a role in prepubertal sex steroid production (Matsumoto et al., 1986Go; Catt and Dufau, 1991Go; Aittomaki et al., 1996Go; Themmen and Huhtaniemi, 2000Go; Barnes et al., 2002Go). Thus sex differences in the control of FSH secretion could serve as a logical avenue for exploration of the cause of differences in timing of puberty. Since FSH secretion is under regulation of not only GnRH but also the FSH-regulatory peptides, we have examined the sex differences in serum concentrations of activin-A, immunoreactive inhibin, inhibin-A and -B, and total follistatin and follistatin 288 in boys and girls.

Inhibin-B concentrations have been shown to be greater in boys than in girls (Crofton et al., 2002aGo,b). Thus it is not surprising that, in this group of children, boys had a 6-fold greater concentration of inhibin-B than did the girls. Immunoreactive inhibin measured in the Monash assay, which measures the inhibin {alpha}-subunit, inhibin precursors, and inhibin-A and -B, exhibits a 2-fold difference between sexes. The increased production of inhibin in boys most probably results in greater negative feedback on FSH compared with girls, contributing to boys’ lower FSH concentrations.

Activin-A increases FSH secretion but has not been shown to vary significantly during changes in human reproductive status, with some exceptions, including variations in the menstrual cycle (Muttukrishna et al., 1996Go) and with ageing (Loria et al., 1998Go; Reame et al., 1998Go). Thus we were surprised to find that activin-A concentrations are significantly greater in girls than in boys, especially in view of the fact that Luisi et al. (2001Go) reported no male–female differences or changes with pubertal advancement. In the study by Luisi et al. (2001Go), activin-A was determined in a single daytime blood sample in children with a wide variety of illnesses including asthma, anaemia and menorrhagia, such that differences in patient population or study protocol might account for the differences in observations regarding activin-A concentration. Nonetheless, the physiological significance of our observed male–female differences in activin-A concentration is unclear. It is believed that virtually all activin in the circulation is bound to follistatin and thus is unlikely to be biologically available (Muttukrishna et al., 1996Go; McConnell et al., 1998Go; Foster et al., 2000Go). Recent studies in sheep have demonstrated that the reduction in FSH that follows follistatin administration is probably due to a reduction in circulating activin availability (Padmanabhan et al., 2002Go). This provides indirect support for an endocrine role for activin. Thus it is conceivable that the greater activin-A concentrations in the serum of girls, in the face of similar follistatin concentrations, may reflect sexual dimorphism in availability of activin, which could contribute to the greater FSH concentrations seen in girls as compared with boys.

We have found previously that total follistatin declines and inhibin-A and -B increase as puberty advances in girls, suggesting that gonadal activation with increased sex steroid production might control follistatin and/or inhibin concentrations (Foster et al., 2000Go). We therefore increased sex steroid concentrations for up to 4 weeks in boys and girls. Despite profound suppression of gonadotrophins, sex steroid treatment did not affect either total follistatin or follistatin 288 concentrations. The pubertal decline in follistatin observed in girls may be regulated by the pubertal increase in GnRH secretion, since GnRH control of follistatin expression has been demonstrated in animal studies (Besecke et al., 1996Go; Bilezikjian et al., 1996Go; Dalkin et al., 1999Go).

Unexpectedly, the increase in sex steroids resulted in a highly significant increase in activin-A in both boys and girls. Activin-A concentrations did not increase in girls with gonadal dysgenesis following estradiol treatment, suggesting that the increase in serum activin-A may be gonadal in origin. Paradoxically, activin-A following sex steroid administration increased in the face of a decline in serum FSH. One would expect that a marked increase in activin concentration would be associated with an increase in FSH secretion unless associated with a tandem increase in follistatin concentration, which did not occur in our subjects. If the change in activin-A seen after sex steroid administration in boys and girls is physiologically relevant, then gonadal secretion of activin-A could be influenced by ambient sex steroid concentrations. Alternatively, the profound suppression of FSH by sex steroids may result in upregulation of activin-A secretion by the gonads as a compensatory mechanism. Neither of these possibilities fits well with the fact that, in our previous study of girls (Foster et al., 2000Go), there was no significant change of activin-A with advancing puberty despite an increase in estradiol concentrations. Thus additional investigations will be needed to assess the physiological relevance of the sex steroid-induced changes in activin-A concentration.

Interestingly, testosterone treatment produced a decline in inhibin-B concentrations in boys, but estradiol failed to do the same in girls. This suggests that the testosterone effects may be mediated via its androgenic action and not via aromatization to estradiol. In contrast, both testosterone and estradiol increased activin-A concentrations in boys and girls, suggesting that the effects of testosterone on activin-A may be facilitated by aromatization to estrogens. Alternatively, the differences may relate to treatment duration. Boys were treated with testosterone for 4 weeks, while nine of the 10 healthy girls were treated for 7 days. The girl who received estradiol for 4 weeks also did not have inhibin-B suppression. FSH does not directly stimulate production of the {beta}B subunit of inhibin-B but does regulate the increase in follicle number (Aittomaki et al., 1996Go). Serum inhibin-B concentrations are proportionate to total follicle mass (Welt et al., 2002Go). If short-term suppression of FSH is insufficient to decrease follicle number, this may explain why estradiol treatment did not reduce inhibin-B concentrations in girls. In boys, either the decline of FSH induced by testosterone or the high concentration of testosterone itself is sufficient to reduce inhibin-B production from the testis.

Our findings suggest that differential production of FSH-regulatory peptides in boys and girls may explain much of the sexual dimorphism in FSH secretion in childhood. Sex steroids play a role in regulating circulating concentrations of activin-A and inhibin-B, but further investigation will be needed to understand the physiological significance of the sex steroid-mediated increase in activin-A concentrations in boys and girls and the decline in inhibin-B concentrations in boys.


    Acknowledgements
 
We gratefully acknowledge Dr Nigel Groome, Oxford Brookes University, Oxford, UK, for provision of follistatin 288 assay reagents. The study was supported by USPHS grant HD16000 and General Clinical Research Center grant M01-RR00042. Presented in part at the 85th Annual Meeting of the Endocrine Society, Philadelphia, PA, June 2003.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aittomaki K, Herva R, Stenman U.H, Juntunen K, Ylostalo P, Hovatta O and de la Chapelle A (1996) Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene. J Clin Endocrinol Metab 81,3722–3726.[Abstract]

Anawalt BD, Bebb RA, Matsumoto AM, Groome NP, Illingworth PJ, McNeilly AS and Bremner WJ (1996) Serum inhibin B levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endocrinol Metab 81,3341–3345.[Abstract]

Andersson A, Juul A, Petersen J, Muller J, Groome N and Skakkebaek N (1997) Serum inhibin B in healthy pubertal and adolescent boys: relation to age, stage of puberty, and follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol levels. J Clin Endocrinol Metab 82,3976–3981.[Abstract/Free Full Text]

Barnes RB, Namnoum AB, Rosenfield RL and Layman LC (2002) The role of LH and FSH in ovarian androgen secretion and ovarian follicular development: clinical studies in a patient with isolated FSH deficiency and multicystic ovaries: case report. Hum Reprod 17,88–91.[Free Full Text]

Bergada I, Rojas G, Ropelato G, Ayuso S, Bergada C and Campo S (1999) Sexual dimorphism in circulating monomeric and dimeric inhibins in normal boys and girls from birth to puberty. Clin Endocrinol 51,455–460.[CrossRef][ISI][Medline]

Besecke LM, Guendner MJ, Schneyer AL, Bauer-Dantoin AC, Jameson JL and Weiss J (1996) Gonadotropin-releasing hormone regulates follicle-stimulating hormone-beta gene expression through an activin/follistatin autocrine or paracrine loop. Endocrinology 137,3667–3673.[Abstract]

Bilezikjian LM, Corrigan AZ, Blount AL and Vale WW (1996) Pituitary follistatin and inhibin subunit messenger ribonucleic acid levels are differentially regulated by local and hormonal factors. Endocrinology 137,4277–4284.[Abstract]

Catt K and Dufau M (1991) Gonadotropic hormones: biosynthesis, secretion, receptors, and actions. In: Yen S and Jaffe R (eds), Reproductive Endocrinology, 3rd edn. WB Saunders, Philadelphia, pp. 105–155.

Cemeroglu AP, Foster CM, Warner R, Kletter GB, Marshall JC and Kelch RP (1996) Comparison of the neuroendocrine control of pubertal maturation in normal girls, in hypogonadal girls, and in boys. J Clin Endocrinol Metab 81,4352–4357.[Abstract]

Crofton PM, Evans AE, Groome NP, Taylor MR, Holland CV and Kelnar CJ (2002a) Dimeric inhibins in girls from birth to adulthood: relationship with age, pubertal stage, FSH and oestradiol. Clin Endocrinol 56,223–230.[CrossRef][ISI][Medline]

Crofton PM, Evans AE, Groome NP, Taylor MR, Holland CV and Kelnar CJ (2002b) Inhibin B in boys from birth to adulthood: relationship with age, pubertal stage, FSH and testosterone. Clin Endocrinol 56,215–221.[CrossRef][ISI][Medline]

Dalkin AC, Haisenleder DJ, Gilrain JT, Aylor K, Yasin M and Marshall JC (1999) Gonadotropin-releasing hormone regulation of gonadotropin subunit gene expression in female rats: actions on follicle-stimulating hormone beta messenger ribonucleic acid (mRNA) involve differential expression of pituitary activin (beta-B) and follistatin mRNAs. Endocrinology 140,903–908[Abstract/Free Full Text]

DePaolo LV, Bicsak TA, Erickson GF, Shimasaki S and Ling N (1991) Follistatin and activin A: a potential intrinsic regulatory system within diverse tissues. Proc Soc Exp Biol Med 198,500–512.[Medline]

deWinter JP, ten Dijke P, de Vries CJ, van Achterberg TA, Sugino H, de Waele P, Huylebroeck D, Verschueren K and van den Eijnden-van Raaij AJ (1996) Follistatins neutralize activin bioactivity by inhibition of activin binding to its type II receptors. Mol Cell Endocrinol 116,105–114.[CrossRef][ISI][Medline]

Evans LW, Muttukrishna S and Groome NP (1998) Development, validation and application of an ultra-sensitive two-site enzyme assay for human follistatin. J Endocrinol 156,275–282.[Abstract/Free Full Text]

Farnsworth PG (1995) Gonadotrophin secretion revisited. How many ways can FSH leave a gonadotroph? J Endocrinol 145,387–395.[Abstract]

Foster CM, Phillips DJ, Wyman T, Evans LW, Groome NP and Padmanabhan V (2000) Changes in serum inhibin, activin and follistatin concentrations during puberty in girls. Hum Reprod 15,1052–1057.[Abstract/Free Full Text]

Greulich WW and Pyle SI (1955) Atlas of Skeletal Development of the Hand and Wrist. Stanford University Press, Stanford, CA.

Grumbach MM and Styne DM (1998) Puberty: ontogeny, neuroendocrinology, physiology, and disorders. In Wilson JD, Foster DW, Kronenberg HM and Larsen PR (eds), Williams Textbook of Endocrinology. WB Saunders, Philadelphia, pp. 1509–1625

Hale PM, Khoury S, Foster CM, Hopwood NJ, Beitins IZ, Marshall JC and Kelch RP (1988) Increased LH pulse frequency during sleep in early to mid-pubertal boys: effects of testosterone infusion. J Clin Endocrinol Metab 66,785–791.[Abstract]

Hayes FJ, Hall JE, Boepple PA and Crowley WF Jr (1998) Differential control of gonadotropin secretion in the human: endocrine role of inhibin. J Clin Endocrinol Metab 83,1835–1841.[Free Full Text]

Knight PG, Muttukrishna S and Groome NP (1996) Development and application of a two-site immunoassay for the determination of ‘total’ activin-A concentrations in serum and follicular fluid. J Endocrinol 148,267–279.[Abstract]

Lee P (1985) Neuroendocrine maturation and puberty. In Lavery J and Sanfilippo J (eds), Pediatric and Adolescent Obstetrics and Gynecology. Springer-Verlag, New York, pp. 12–26.

Loria P, Petraglia F, Concari M, Bertolotti M, Martella P, Luisi S, Grisolia C, Foresta C, Volpe A, Genazzani AR and Carulli N (1998) Influence of age and sex on serum concentrations of total dimeric activin A. Eur J Endocrinol 139,487–492.[ISI][Medline]

Luisi S, Lombardi I, Florio P, Cobellis L, Iughetti L, Bernasconi S, Genazzani AR, Petraglia F (2001) Serum activin A levels in males and females during pubertal development. Gynecol Endocrinol 15,1–4.[ISI][Medline]

Marshall JC and Kelch RP (1986) Gonadotropin-releasing hormone: role of pulsatile secretion in the regulation of reproduction. N Engl J Med 315,1459–1468.[ISI][Medline]

Mather JP, Woodruff TK and Krummen LA (1992) Paracrine regulation of reproductive function by inhibin and activin. Proc Soc Exp Biol Med 201,1–15.[Medline]

Matsumoto AM, Karpas AE and Bremner WJ (1986) Chronic human chorionic gonadotropin administration in normal men: evidence that follicle-stimulating hormone is necessary for the maintenance of quantitatively normal spermatogenesis in man. J Clin Endocrinol Metab 62,1184–1192.[Abstract]

McConnell DS, Wang Q, Sluss PM, Bolf N, Khoury RH, Schneyer AL, Midgley AR Jr, Reame NE, Crowley WF Jr and Padmanabhan V (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 83,851–858[Abstract/Free Full Text]

McNeilly AS (1988) The control of FSH secretion. Acta Endocrinol Suppl 288,31–40

Muttukrishna S, Fowler PA, George L, Groome NP and Knight PG (1996) Changes in peripheral serum levels of total activin-A during the human menstrual cycle and pregnancy. J Clin Endocrinol Metab 81,3328–3334.[Abstract]

Muyan M, Ryzmkiewicz DM and Boime I (1994) Secretion of lutropin and follitropin from transfected GH3 cells: evidence for separate secretory pathways. Mol Endocrinol 8,1789–1797.[Abstract]

O’Connor AE, McFarlane JR, Hayward S, Yohkaichiya T Groome NP and de Kretser DM (1999) Serum activin A and follistatin concentrations during human pregnancy: a cross sectional and longitudinal study. Hum Reprod 14,827–832.[Abstract/Free Full Text]

Padmanabhan V and Sharma T (2001) Neuroendocrine versus paracrine control of follicle-stimulating hormone. Arch Med Res 32,533–543.[CrossRef][ISI][Medline]

Padmanabhan V and West C (2001) Endocrine, autocrine, and paracrine actions of inhibin/activin/follistatin on follicle-stimulating hormone. In Muttukrishna S and Ledger W (eds), Inhibin, Activin and Follistatin in Human Reproductive Physiology. Imperial College Press, London, UK, pp. 61–90.

Padmanabhan V, Battaglia D, Brown MB, Karsch FJ, Lee JS, Pan W, Phillips DJ and VanCleeff J (2002) Neuroendocrine control of follicle-stimulating hormone secretion: II. Is follistatin-induced suppression of follicle-stimulating hormone secretion mediated via changes in activin availability and does it involve changes in GnRH secretion? Biol Reprod 66,1395–1402.[Abstract/Free Full Text]

Phillips DJ and de Kretser DM (1998) Follistatin: a multifunctional regulatory protein. Front Neuroendocrinol 19,287–322.[CrossRef][ISI][Medline]

Reame NE, Wyman TL, Phillips DJ, de Kretser DM and Padmanabhan V (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 83,3302–3307.[Abstract/Free Full Text]

Roberts V, Barth S, el-Roeiy A and Yen S (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 77,1402–1410.[Abstract]

Robertson D (1992) Follistatin/activin-binding protein. Trends Endocrinol Metab 3,65–68.[ISI]

Robertson DM, Hayward S, Irby D, Jacobsen J, Clarke L, McLachlan RI, de Kretser DM (1988) Radioimmunoassay of rat serum inhibin: changes after PMSG stimulation and gonadectomy. Mol Cell Endocrinol 58,1–8.[CrossRef][ISI][Medline]

Robertson DM, Giacometti, M, Foulds LM, Lahnstein J, Goss NH, Hearn MT and de Kretser DM (1989) Isolation of inhibin alpha-subunit precursor proteins from bovine follicular fluid. Endocrinology 125,2141–2149.[Abstract]

Sehested A, Juul AA, Andersson AM, Petersen JH, Jensen TK, Muller J, Skakkebaek NE (2000) Serum inhibin A and inhibin B in healthy prepubertal, pubertal, and adolescent girls and adult women: relation to age, stage of puberty, menstrual cycle, follicle-stimulating hormone, luteinizing hormone, and estradiol levels. J Clin Endocrinol Metab 85,1634–1640.[Abstract/Free Full Text]

Tanner JM (1978) Growth at Adolescence. Blackwell, Oxford.

Themmen APN and Huhtaniemi IT (2000) Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary–gonadal function. Endocr Rev 21,551–583.[Abstract/Free Full Text]

Welt C, Sidis Y, Keutmann H and Schneyer A (2002) Activins, inhibins, and follistatins: from endocrinology to signalling. A paradigm for the new millennium. Exp Biol Med 227,724–752.[Abstract/Free Full Text]

Winter JS (1982) Hypothalamic–pituitary function in the fetus and infant. Clin Endocrinol Metab 11,41–55.[ISI][Medline]

Ying S-Y (1988) Inhibins, activins and follistatins: regulatory proteins modulating the secretion of follicle-stimulating hormone. Endocr Rev 9, 267–293.[Abstract]

Submitted on January 29, 2004; accepted on March 31, 2004.