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
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Abstract |
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Key words: activin/follistatin/FSH/inhibin/puberty
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Introduction |
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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, 1988; Ying, 1988
; Muyan et al., 1994
; Farnsworth, 1995
; Padmanabhan and Sharma, 2001
; Padmanabhan and West, 2001
). These peptides include the activins and inhibins, members of the transforming growth factor-
(TGF-
) family, and the follistatins. The activins stimulate FSH secretion (Ying, 1988
) and are made in the gonads, pituitary and extragonadal tissue (DePaolo et al., 1991
; Mather et al., 1992
). Activin-A, comprised of two
A subunits, is the only activin peptide for which relevant serum measurements have been available (Knight et al., 1996
). 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., 1998
; Reame et al., 1998
) and, in some studies, during the menstrual cycle (Muttukrishna et al., 1996
). Activin-A concentrations have been reported to remain stable during pubertal maturation in boys and girls (Foster et al., 2000
; Luisi et al., 2001
).
Inhibins suppress FSH secretion (Ying, 1988; Welt et al., 2002
). They are structurally related to the activins but contain an
-subunit in combination with an activin
-subunit (Ying, 1988
). Inhibin-A and inhibin-B are both produced in the ovaries, while the testis produces only inhibin-B (Roberts et al., 1993
; Anawalt et al., 1996
; Hayes et al., 1998
). 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., 1999
; Foster et al., 2000
; Sehested et al., 2000
). Inhibin-A is produced principally in dominant ovarian follicles (Roberts et al., 1993
; Welt et al., 2002
). 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. 2000
; Sehested et al., 2000
; Crofton et al., 2002a
). Inhibin-B is associated with small antral follicles (Roberts et al., 1993
; Welt et al., 2002
). 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., 1997
; Crofton et al., 2002b
).
The third FSH regulator, follistatin, is a monomeric peptide that binds activin and prevents activin binding to its receptor, thereby inhibiting FSH secretion (Robertson, 1992; de Winter et al., 1996
; Phillips and deKretser, 1998
). Processing of mRNA results in at least two forms of follistatin, a 315 kDa peptide and a 288 kDa peptide (Robertson, 1992
; Phillips and deKretser, 1998
). Follistatins are made in the gonads, pituitary and extragonadal tissues (Robertson, 1992
; Phillips and deKretser, 1998
).
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., 2000). 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.
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Materials and methods |
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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: 20.0022.45 h; 23.0001.45 h; 02.0004.45 h; and 05.0008.00 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., 1998). 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., 1988) using an in-house human recombinant (hr) inhibin-A as both standard and tracer. The assay cross-reacts 288% with pro-
C, the pro-sequence of the inhibin
-subunit (Robertson et al., 1989
). 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 (OConnor et al., 1999) 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 Students t-test. A P-value of <0.05 was considered to be significant.
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Results |
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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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Discussion |
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Inhibin-B concentrations have been shown to be greater in boys than in girls (Crofton et al., 2002a,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
-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., 1996) and with ageing (Loria et al., 1998
; Reame et al., 1998
). 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. (2001
) reported no malefemale differences or changes with pubertal advancement. In the study by Luisi et al. (2001
), 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 malefemale 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., 1996
; McConnell et al., 1998
; Foster et al., 2000
). 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., 2002
). 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., 2000). 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., 1996
; Bilezikjian et al., 1996
; Dalkin et al., 1999
).
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., 2000), 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 B subunit of inhibin-B but does regulate the increase in follicle number (Aittomaki et al., 1996
). Serum inhibin-B concentrations are proportionate to total follicle mass (Welt et al., 2002
). 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.
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Acknowledgements |
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Submitted on January 29, 2004; accepted on March 31, 2004.