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The role of estrogen in bone growth and maturation during childhood and adolescence
J. SteroidBiochem. Molec. Biol. Vol. 61, No. 3-6, pp. 141-144, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain
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T h e Role o f E s t r o g e n in B o n e G r o w t h and M a t u r a t i o n D u r i n g C h i l d h o o d and Adolescence G. B. Cutler Jr* Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, Bethesda, MD 20892-1862, U.S.A.
T w e n t y y e a r s ago it was be l i e ve d t h a t p u b e r t a l grow t h was s t i m u l a t e d by t e s t i c u l a r a n d r o g e n in boys a n d b y a d r e n a l a n d r o g e n in girls. E s t r o g e n , w hi ch was used to inhibit grow t h in excessively tall girls, was n o t t h o u g h t to have g r o w t h - p r o m o t i n g effects. We h y p o t h e s i z e d t h a t e s t r o g e n has a b i p h a sic effect on e p i p h y s e a l growth, with m a x i m a l s t i m u l a t i o n at low levels. We show ed t h a t t he a d m i n i s t r a t i o n o f low doses o f e s t r o g e n , c o r r e s p o n d i n g to a s e r u m est radi ol level o f a b o u t 4 p g / m l (15 pmol/l) c a u s e d m o r e t h a n a 60% i n c r e a s e o v e r the p r e p u b e r t a l grow t h r a t e in b o t h boys a n d girls. To test th e h y p o t h e s i s t h a t e s t r o g e n is t he p r i n c i p a l m e d i a t o r o f the p u b e r t a l grow t h s p u r t in boys, we a d m i n i s t e r e d t he a r o m a t a s e i n h i b i t o r , t e s t o l a c t o n e , to boys with familial m a l e - l i m i t e d p r e cocious p u b e r t y . T e s t o l a c t o n e p r o d u c e d n e a r n o r m a l i z a t i o n o f b o t h g r o w t h velocity a n d b o n e m a t u r a t i o n , despite levels o f s e r u m t e s t o s t e r o n e t h a t r e m a i n e d within the adul t m a l e f a n g e . T h e o b s e r v a t i o n t h a t low levels o f e s t r o g e n s t i m u l a t e g r o w t h a n d b o n e m a t u r a t i o n suggested t h a t e stro gen m i g h t explain t he m o r e r a p i d e p i p h y s e a l m a t u r a t i o n o f p r e p u b e r t a l girls c o m p a r e d to boys. T o d e t e r m i n e w h e t h e r p r e p u b e r t a l girls h a v e h i g h e r e s t r o g e n levels t h a n p r e p u b e r t a l boys, we develo p e d an u l t r a s e n s i t i v e r e c o m b i n a n t cell b i o a s s a y for e s t r o g e n with a sensitivity o f 0.02 pg/ml (0.07 pmol/1) es t r adi ol equivalents. P r e p u b e r t a l girls h a d a p p r o x i m a t e l y eight-fold h i g h e r levels o f s e r u m e s t r a d i o l t h a n did p r e p u b e r t a l boys (0.6 + 0.6 pg/ml (SD) (2.2 + 2.2 pmol/1) vs 0.08 + 0.2 pg/ml (0.29 _+ 0.73 pmol/1), P < 0.05). We c o n c l u d e d t h a t t he p u b e r t a l grow t h s p u r t o f b o t h sexes is d r i v e n p r i m a r i l y b y e s t r o g e n , a n d t h a t the m o r e r a p i d e p i p h y s e a l m a t u r a ü o n o f p r e p u b e r t a l girls (vs boys) m a y be e x p l a i n e d b y t h e i r h i g h e r e s t r a d i o l levels. © 1997 E l sevi er Sci ence L t d
J. SteroidBiochem. Molec. Biol., Vol. 61, No. 3-6, pp. 141-144, 1997
INTRODUCTION T h e human pubertal growth spurt is a two-edged sword. On the one hand, it doubles the prepubertal growth rate and contributes more than 15% to the total adult height. On the other hand, it initiates epiphyseal fusion, which terminates linear growth. This review will address the following questions about the role of estrogen in both prepubertal and pubertal growth:
2. What is the dose-response relationship between estradiol level and linear growth? 3. Which hormone(s) cause epiphyseal fusion? 4. Does endogenous estrogen stimulate bone maturation in prepubertal girls?
R O L E O F E S T R O G E N IN P U B E R T A L G R O W T H
Twenty years ago, the pubertal growth spurt was thought to be driven by testicular androgen in boys 1. Which hormone(s) cause the pubertal growth and by adrenal androgen in girls [1]. However, conspurt? siderable clinical evidence pointed against adrenal androgen playing a role in pubertal growth, because Proceedings of the IV International Aromatase Conference, Tahoe the pubertal growth spurt could be dissociated from City, CA, U.S.A., 7-11 June 1996. *Correspondence to G. B. Cutler, Jr. Tel: +1 301 496 4686; the adrenarche in numerous disorders, including Fax: +1 301 402 0574; e-mail: [email protected]. childhood Addison's disease, precocious puberty, 141
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premature adrenarche, Kallman's syndrome and T u r n e r ' s syndrome. We therefore hypothesized that estrogen was driving the pubertal growth spurt in girls, and in addition that it m u s t be doing so at low levels. T h e reason that low levels of estrogen were proposed to stimulate growth was that the onset of the pubertal growth spurt in girls precedes breast budding by up to 6 months. However, doses of estrogen similar to or slightly higher than those used for estrogen replacement in adult w o m e n had been shown to inhibit growth rate in adolescent girls with tall stature. T a k e n together, these observations suggested the hypothesis that estrogen has a biphasic dose-response relationship for epiphyseal growth, with stimulation at low levels and a reduced effect, or even inhibition, at high levels. Dose-response studies in girls with T u r n e r ' s syndrome confirmed the biphasic effect of estrogen on linear growth [2, 3]. T h e greatest increase in growth velocity occurred at an ethinyl estradiol dose of 100 ng/kg/day, which represented a daily dose of approximately 4/~g for a typical 40 kg girl. T h e higher doses of 400 and 800 ng/kg/day did not increase the growth rate. These observations suggest that feminization of girls with T u r n e r ' s syndrome should begin with a period of low-dose estrogen replacement to p r o m o t e accelerated growth with the least acceleration of bone age. O u t current r e c o m m e n d a t i o n is that ethinyl estradiol be initiated at 5 0 - 1 0 0 ng/kg/day, with a dose reduction to 25 ng/kg/day in some girls in w h o m breast development, accelerated bone maturation, or menarche suggest particular sensitivity. T h e optimal age for initiating estrogen therapy remains controversial, but is probably between 12 and 14 years when the overall welfare of the child, including psychological measures, is taken into account [4]. We next asked whether estrogen also influences the growth of boys with a similar dose-response curve. T o answer this question, three doses of estradiol (4, 20 and 90/~g/day) were infused intravenously for 4 days in boys with idiopathic delay of puberty [5]. Ulnar growth rate was determined over 3-week periods that began with each estradiol infusion. As in the girls with T u r n e r ' s syndrome, the greatest growth-promoting effect (an approximate tripling of the baseline growth rate) occurred at the dose of 4/~g estradiol per day. Although the estradiol level corresponding to this dose could not be measured accurately because it was too near the assay detection limit, it was estimated to be 4 pg/ml (15 pmol/1) by extrapolation of the levels at the 20/~g/day and 90/~g/day doses. We concluded from this study that low-dose estradiol can stimulate ulnar growth in boys, that the optimal growth-promoting level of estradiol is approximately 4 pg/ml (15 pmol/1), and that estradiol m a y play a role in the male pubertal growth spurt.
Additional evidence that estrogen plays a role in mediating male pubertal growth came from studies of familial male-limited precocious puberty. This rare disorder results from activating mutations of the luteinizing h o r m o n e receptor, which cause adult levels of circulating testosterone by 2 - 3 years of age [6, 7]. Administration of the aromatase inhibitor, testolactone, caused a marked reduction of both growth velocity and bone maturation rate to nearly normal prepubertal levels [8]. Administration of the antiandrogen, spironolactone, was less effective in decreasing growth rate, and had no apparent effect on the markedly elevated rate of bone maturation. T h e s e observations support the hypothesis that estrogen, rather than androgen, is the major mediator of accelerated growth rate and bone maturation in pubertal boys.
R O L E O F E S T R O G E N IN E P I P H Y S E A L FUSION
Recently two dramatic experiments of nature have produced a paradigm shift in our understanding of the relative roles played by estrogen and androgen in mediating epiphyseal fusion in men. First, Smith et al. [9] discovered a 28-year-old m a n with complete estrogen resistance because of a homozygous non-sense mutation in the estrogen receptor gene [10]. Despite normal virilization and adult male testosterone levels for more than a decade after the completion of p u b erty, this 28-year-old m a n had unfused epiphyses, a bone age of 15, and a height of 6 ft 8 ins (204 cm). Second, Morishima et al. [11] discovered a 24-yearold m a n with aromatase deficiency because of a homozygous reis-sense mutation in the aromatase gene. Despite full sexual maturity and supraphysiological testosterone levels (2015 ng/dl (70 nmol/1)), this 24-year-old m a n had unfused epiphyses, a bone age of 14, and a height of 6 f t 8ins (204 cm). Interestingly, neither of these m e n had a d o c u m e n t e d pubertal growth spurt, hut instead attained their extraordinary stature simply by continuing to grow long after normal males have undergone epiphyseal fusion. T h e s e remarkable cases provide compelling evidence that androgen alone, even at levels above the normal adult male fange, does not fuse the epiphyses. Rather, it is estrogen that fuses the epiphyses and terminates linear growth.
DOES ESTROGEN STIMULATE BONE M A T U R A T I O N IN P R E P U B E R T A L GIRLS?
T h e epiphyses of prepubertal girls mature approximately 20% faster than the epiphyses of prepubertal boys, and therefore gender-specific standards must be used to determine bone age [12]. W h a t drives this more rapid epiphyseal maturation of prepubertal girls?
Estrogen and Bone Development We hypothesized that prepubertal girls have higher estradiol levels than prepubertal boys, and that these higher estradiol levels cause their more rapid skeletal maturation. A review of previous studies did not support the hypothesis of higher estradiol levels in prepubertal girls [13]. However, the accuracy of these observations was questionable because the observed estradiol levels were close to the assay detection limit. T o re-examine this issue, we developed an ultrasensitive recombinant cell bioassay for serum estradiol [14]. T h e assay employed a yeast strain that was genetically engineered for extreme sensitivity to estrogen. Yeasts were transformed with plasmids encoding the h u m a n estrogen receptor and a reporter system consisting of the fl-galactosidase structural gene fused to an estrogen-responsive promoter. Dried ether extracts of 0.8 ml serum were incubated with the transformed yeast cells for 8 h, after which the fl-galactosidase activity was measured. T h e assay detection limit for serum estradiol was 0.02 pg/ml (0.07pmol/1). T h e assay was unexpectedly specific for estradiol, with approximately 0.3% crossreactivity with estrone and estriol. At serum estradiol levels ranging from 15 to 300pg/ml (55-1100pmol/1), the correlation coefficient between estradiol levels by the recombinant cell bioassay and an existing estradiol radioimmunoassay was 0.90. With this new assay, the mean serum estradiol level in 21 prepubertal girls (aged 7.5 ___2.1 yr (SD)) was 0.6 + 0.6 pg/ml (2.2 + 2.2 pmol/1), which was significantly greater ( P < 0.05) than the level in 23 prepubertal boys (9.5 _+ 2.1 yr): 0.08 __+0.2 pg/ml (0.29 + 0.73 pmol/1). Additionally, the levels in both sexes were far lower than those that had been determined previously by radioimmunoassay [13]. Prepubertal girls have thus approximately eight-fold higher estradiol levels than prepubertal boys, which may explain their more rapid epiphyseal maturation. These observations allow us to infer the dose-response relationship between circulating estradiol levels and the growth rate and bone maturation of children and adolescents. Estradiol levels observed in prepubertal boys (0.08pg/ml (0.3 pmol/1)) appear to be without effect on growth rate or bone maturation. We hypothesize that estradiol levels in prepubertal girls (0.6pg/ml (2.2 pmol/1)) cause the 20% more rapid epiphyseal maturation that is observed in girls compared to boys. T h e absence of this estradiol in girls with Turner's syndrome may explain their corresponding delay in bone maturation compared to normal girls [15]. Still higher levels of estradiol begin to exert major effects on growth velocity, with the maximal growth rate during male puberty corresponding to estradiol levels of approximately 3 - 4 pg/ml (1115 pmol/1) [16]. Further increases in estradiol level, to 12 pg/ml (44 pmol/1) and above, progressively appear
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to uncouple growth velocity from epiphyseal maturation and fusion. In conclusion, pubertal growth and skeletal maturation in both sexes is driven primarily by estrogen. T h e dose-response curve for the growth-promoting action of estrogen is biphasic, with maximal stimulation of growth at estradiol levels of approximately 3 - 4 pg/ml (11-15 pmol/1). Androgen at or above normal adult male levels does not fuse the epiphyses. Rather, it is estrogen that fuses the epiphyses and terminates linear growth. Lastly, prepubertal girls have eight-fold higher estradiol levels than prepubertal boys, which may explain their more rapid epiphyseal maturation.
REFERENCES 1. Cutler G. B. Jr, Cassorla F. C., Ross J. L., Pescovitz O. H., Barnes K. M., Comite F., Feuillan P. P., Laue L., Foster C. M., Kenigsberg D., Caruso-Nicoletti M., Garcia H. B., Urairte M. M., Hench K. D., Skerda M. C., Long L. M. and Loriaux D. L.: Pubertal growth: physiology and pathophysiology. Recent Progr. Horm. Res. 42 (1986) 443-470. 2. Ross J. L., Cassorla F. G., Skerda M. C., Valk I. M., Loriaux D. L. and Cutler G. B. Jr: A preliminary study of the effect of estrogen dose on growth in Turner's syndrome. N. Engl. J. Med. 309 (1983) 1104-1106. 3. Ross J. L., Long L. M., Skerda M. C., Cassorla F. G., Loriaux D. L. and Cufler G. B. Jr: Effect of low doses of estradiol on six month growth rates and predicted height in patients with Turner syndrome. J. Pediatr. 109 (1986) 950-953. 4. Ross J. L., McCauley E., Roeltgen D., Long L., Kushner H., FeuiUan P. and Cutler G. B. Jr: Self-concept and behavior in adolescent girls with Turner syndrome: potential estrogen effects. J. Clin. Endocr. Metab. 81 (1996) 926-931. 5. Caruso-Nicoletti M., Cassorla F. G., Skerda M. C., Ross J. L., Loriaux D. L. and Cutler G. B. Jr: Short term, low dose estradiol accelerates ulnar growth in boys. J. Clin. Endocr. Metab. 61 (1985) 896-898. 6. Shenker A., Laue L., Kosugi S., Merendino J. J. Jr, Minegishi T. and Cutler G. B. Jr: A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365 (1993) 652-654. 7. Laue L., Chan W. Y., Hsueh A., Wu S. M., Blomberg L. and Cutler G. B. Jr: Genetic heterogeneity of constitutively activating mutations of the luteinizing hormone receptor in familial male-limited precocious puberty. Proc. Nam. Acad. Sci. U.S.A. 92 (1995) 1906-1910. 8. Laue L., Kenigsberg D., Pescovitz O., Hench K. D., Barnes K. M., Loriaux D. L. and Cutler G. B. Jr: Treatment of familial male precocious puberty with spironolactone and testolactone. N. Engl. J. Med. 320 (1989) 496-502. 9. Smith E. P., Boyd J., Frank G. R., Takahashi H., Cohen R. M., Specker B., Williams T. C., Lubahn D. B. and Korach K. S.: Estrogen resistance caused by a mutation in the estrogenreceptor gene in a man. N. Engl. J. Med. 331 (1994) 10561061. 10. Federman D. D.: Life without estrogen. N. Engl. J. Med. 331 (1994) 1088-1089. 11. Morishima A., Grumbach M. M., Simpson E. R., Fisher C. and Qin K.: Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J. Clin. Endocr. Metab. 80 (1995) 36893698. 12. Greulich W. W. and Pyle S. I.: Radiographic Atlas of Skeletal Development of the Hand and Wrist. Stanford University Press, Stanford, CA, 1959.
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13. Bidlingmaier F., Wagner-Bamack M., Butenandt O. and Knorr D.: Plasma estrogens in childhood and puberty under physiologic and pathologic conditions. Pediatr. Res. 7 (1973) 901907. 14. Klein K. O., Baron J., Colli M., McDonnell D. P. and Cutler G. B. Jr: Estrogen levels in childhood determined by a new ultrasensitive recombinant cell bioassay, fr. Clin. Invest. 94 (1994) 2475-2480.
15. Midtbo M. and Halse A.: Skeletal maturity, dental maturity, and eruption in young patients with Turner syndrome. Acta OdontoL Scand. 50 (1992) 303-312. 16. Klein K. O., Martha P. M. Jr, Blizzard R. M., Herbst T. and Rogol A. D.: A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. II. Estrogen levels as determined by an ultrasensitive bioassay..7. Clin. Endocrinol. Metab. 81 (1996) 3203-3207.
Pergamon
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T h e Role o f E s t r o g e n in B o n e G r o w t h and M a t u r a t i o n D u r i n g C h i l d h o o d and Adolescence G. B. Cutler Jr* Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, Bethesda, MD 20892-1862, U.S.A.
T w e n t y y e a r s ago it was be l i e ve d t h a t p u b e r t a l grow t h was s t i m u l a t e d by t e s t i c u l a r a n d r o g e n in boys a n d b y a d r e n a l a n d r o g e n in girls. E s t r o g e n , w hi ch was used to inhibit grow t h in excessively tall girls, was n o t t h o u g h t to have g r o w t h - p r o m o t i n g effects. We h y p o t h e s i z e d t h a t e s t r o g e n has a b i p h a sic effect on e p i p h y s e a l growth, with m a x i m a l s t i m u l a t i o n at low levels. We show ed t h a t t he a d m i n i s t r a t i o n o f low doses o f e s t r o g e n , c o r r e s p o n d i n g to a s e r u m est radi ol level o f a b o u t 4 p g / m l (15 pmol/l) c a u s e d m o r e t h a n a 60% i n c r e a s e o v e r the p r e p u b e r t a l grow t h r a t e in b o t h boys a n d girls. To test th e h y p o t h e s i s t h a t e s t r o g e n is t he p r i n c i p a l m e d i a t o r o f the p u b e r t a l grow t h s p u r t in boys, we a d m i n i s t e r e d t he a r o m a t a s e i n h i b i t o r , t e s t o l a c t o n e , to boys with familial m a l e - l i m i t e d p r e cocious p u b e r t y . T e s t o l a c t o n e p r o d u c e d n e a r n o r m a l i z a t i o n o f b o t h g r o w t h velocity a n d b o n e m a t u r a t i o n , despite levels o f s e r u m t e s t o s t e r o n e t h a t r e m a i n e d within the adul t m a l e f a n g e . T h e o b s e r v a t i o n t h a t low levels o f e s t r o g e n s t i m u l a t e g r o w t h a n d b o n e m a t u r a t i o n suggested t h a t e stro gen m i g h t explain t he m o r e r a p i d e p i p h y s e a l m a t u r a t i o n o f p r e p u b e r t a l girls c o m p a r e d to boys. T o d e t e r m i n e w h e t h e r p r e p u b e r t a l girls h a v e h i g h e r e s t r o g e n levels t h a n p r e p u b e r t a l boys, we develo p e d an u l t r a s e n s i t i v e r e c o m b i n a n t cell b i o a s s a y for e s t r o g e n with a sensitivity o f 0.02 pg/ml (0.07 pmol/1) es t r adi ol equivalents. P r e p u b e r t a l girls h a d a p p r o x i m a t e l y eight-fold h i g h e r levels o f s e r u m e s t r a d i o l t h a n did p r e p u b e r t a l boys (0.6 + 0.6 pg/ml (SD) (2.2 + 2.2 pmol/1) vs 0.08 + 0.2 pg/ml (0.29 _+ 0.73 pmol/1), P < 0.05). We c o n c l u d e d t h a t t he p u b e r t a l grow t h s p u r t o f b o t h sexes is d r i v e n p r i m a r i l y b y e s t r o g e n , a n d t h a t the m o r e r a p i d e p i p h y s e a l m a t u r a ü o n o f p r e p u b e r t a l girls (vs boys) m a y be e x p l a i n e d b y t h e i r h i g h e r e s t r a d i o l levels. © 1997 E l sevi er Sci ence L t d
J. SteroidBiochem. Molec. Biol., Vol. 61, No. 3-6, pp. 141-144, 1997
INTRODUCTION T h e human pubertal growth spurt is a two-edged sword. On the one hand, it doubles the prepubertal growth rate and contributes more than 15% to the total adult height. On the other hand, it initiates epiphyseal fusion, which terminates linear growth. This review will address the following questions about the role of estrogen in both prepubertal and pubertal growth:
2. What is the dose-response relationship between estradiol level and linear growth? 3. Which hormone(s) cause epiphyseal fusion? 4. Does endogenous estrogen stimulate bone maturation in prepubertal girls?
R O L E O F E S T R O G E N IN P U B E R T A L G R O W T H
Twenty years ago, the pubertal growth spurt was thought to be driven by testicular androgen in boys 1. Which hormone(s) cause the pubertal growth and by adrenal androgen in girls [1]. However, conspurt? siderable clinical evidence pointed against adrenal androgen playing a role in pubertal growth, because Proceedings of the IV International Aromatase Conference, Tahoe the pubertal growth spurt could be dissociated from City, CA, U.S.A., 7-11 June 1996. *Correspondence to G. B. Cutler, Jr. Tel: +1 301 496 4686; the adrenarche in numerous disorders, including Fax: +1 301 402 0574; e-mail: [email protected]. childhood Addison's disease, precocious puberty, 141
142
G.B. Cutler Jr.
premature adrenarche, Kallman's syndrome and T u r n e r ' s syndrome. We therefore hypothesized that estrogen was driving the pubertal growth spurt in girls, and in addition that it m u s t be doing so at low levels. T h e reason that low levels of estrogen were proposed to stimulate growth was that the onset of the pubertal growth spurt in girls precedes breast budding by up to 6 months. However, doses of estrogen similar to or slightly higher than those used for estrogen replacement in adult w o m e n had been shown to inhibit growth rate in adolescent girls with tall stature. T a k e n together, these observations suggested the hypothesis that estrogen has a biphasic dose-response relationship for epiphyseal growth, with stimulation at low levels and a reduced effect, or even inhibition, at high levels. Dose-response studies in girls with T u r n e r ' s syndrome confirmed the biphasic effect of estrogen on linear growth [2, 3]. T h e greatest increase in growth velocity occurred at an ethinyl estradiol dose of 100 ng/kg/day, which represented a daily dose of approximately 4/~g for a typical 40 kg girl. T h e higher doses of 400 and 800 ng/kg/day did not increase the growth rate. These observations suggest that feminization of girls with T u r n e r ' s syndrome should begin with a period of low-dose estrogen replacement to p r o m o t e accelerated growth with the least acceleration of bone age. O u t current r e c o m m e n d a t i o n is that ethinyl estradiol be initiated at 5 0 - 1 0 0 ng/kg/day, with a dose reduction to 25 ng/kg/day in some girls in w h o m breast development, accelerated bone maturation, or menarche suggest particular sensitivity. T h e optimal age for initiating estrogen therapy remains controversial, but is probably between 12 and 14 years when the overall welfare of the child, including psychological measures, is taken into account [4]. We next asked whether estrogen also influences the growth of boys with a similar dose-response curve. T o answer this question, three doses of estradiol (4, 20 and 90/~g/day) were infused intravenously for 4 days in boys with idiopathic delay of puberty [5]. Ulnar growth rate was determined over 3-week periods that began with each estradiol infusion. As in the girls with T u r n e r ' s syndrome, the greatest growth-promoting effect (an approximate tripling of the baseline growth rate) occurred at the dose of 4/~g estradiol per day. Although the estradiol level corresponding to this dose could not be measured accurately because it was too near the assay detection limit, it was estimated to be 4 pg/ml (15 pmol/1) by extrapolation of the levels at the 20/~g/day and 90/~g/day doses. We concluded from this study that low-dose estradiol can stimulate ulnar growth in boys, that the optimal growth-promoting level of estradiol is approximately 4 pg/ml (15 pmol/1), and that estradiol m a y play a role in the male pubertal growth spurt.
Additional evidence that estrogen plays a role in mediating male pubertal growth came from studies of familial male-limited precocious puberty. This rare disorder results from activating mutations of the luteinizing h o r m o n e receptor, which cause adult levels of circulating testosterone by 2 - 3 years of age [6, 7]. Administration of the aromatase inhibitor, testolactone, caused a marked reduction of both growth velocity and bone maturation rate to nearly normal prepubertal levels [8]. Administration of the antiandrogen, spironolactone, was less effective in decreasing growth rate, and had no apparent effect on the markedly elevated rate of bone maturation. T h e s e observations support the hypothesis that estrogen, rather than androgen, is the major mediator of accelerated growth rate and bone maturation in pubertal boys.
R O L E O F E S T R O G E N IN E P I P H Y S E A L FUSION
Recently two dramatic experiments of nature have produced a paradigm shift in our understanding of the relative roles played by estrogen and androgen in mediating epiphyseal fusion in men. First, Smith et al. [9] discovered a 28-year-old m a n with complete estrogen resistance because of a homozygous non-sense mutation in the estrogen receptor gene [10]. Despite normal virilization and adult male testosterone levels for more than a decade after the completion of p u b erty, this 28-year-old m a n had unfused epiphyses, a bone age of 15, and a height of 6 ft 8 ins (204 cm). Second, Morishima et al. [11] discovered a 24-yearold m a n with aromatase deficiency because of a homozygous reis-sense mutation in the aromatase gene. Despite full sexual maturity and supraphysiological testosterone levels (2015 ng/dl (70 nmol/1)), this 24-year-old m a n had unfused epiphyses, a bone age of 14, and a height of 6 f t 8ins (204 cm). Interestingly, neither of these m e n had a d o c u m e n t e d pubertal growth spurt, hut instead attained their extraordinary stature simply by continuing to grow long after normal males have undergone epiphyseal fusion. T h e s e remarkable cases provide compelling evidence that androgen alone, even at levels above the normal adult male fange, does not fuse the epiphyses. Rather, it is estrogen that fuses the epiphyses and terminates linear growth.
DOES ESTROGEN STIMULATE BONE M A T U R A T I O N IN P R E P U B E R T A L GIRLS?
T h e epiphyses of prepubertal girls mature approximately 20% faster than the epiphyses of prepubertal boys, and therefore gender-specific standards must be used to determine bone age [12]. W h a t drives this more rapid epiphyseal maturation of prepubertal girls?
Estrogen and Bone Development We hypothesized that prepubertal girls have higher estradiol levels than prepubertal boys, and that these higher estradiol levels cause their more rapid skeletal maturation. A review of previous studies did not support the hypothesis of higher estradiol levels in prepubertal girls [13]. However, the accuracy of these observations was questionable because the observed estradiol levels were close to the assay detection limit. T o re-examine this issue, we developed an ultrasensitive recombinant cell bioassay for serum estradiol [14]. T h e assay employed a yeast strain that was genetically engineered for extreme sensitivity to estrogen. Yeasts were transformed with plasmids encoding the h u m a n estrogen receptor and a reporter system consisting of the fl-galactosidase structural gene fused to an estrogen-responsive promoter. Dried ether extracts of 0.8 ml serum were incubated with the transformed yeast cells for 8 h, after which the fl-galactosidase activity was measured. T h e assay detection limit for serum estradiol was 0.02 pg/ml (0.07pmol/1). T h e assay was unexpectedly specific for estradiol, with approximately 0.3% crossreactivity with estrone and estriol. At serum estradiol levels ranging from 15 to 300pg/ml (55-1100pmol/1), the correlation coefficient between estradiol levels by the recombinant cell bioassay and an existing estradiol radioimmunoassay was 0.90. With this new assay, the mean serum estradiol level in 21 prepubertal girls (aged 7.5 ___2.1 yr (SD)) was 0.6 + 0.6 pg/ml (2.2 + 2.2 pmol/1), which was significantly greater ( P < 0.05) than the level in 23 prepubertal boys (9.5 _+ 2.1 yr): 0.08 __+0.2 pg/ml (0.29 + 0.73 pmol/1). Additionally, the levels in both sexes were far lower than those that had been determined previously by radioimmunoassay [13]. Prepubertal girls have thus approximately eight-fold higher estradiol levels than prepubertal boys, which may explain their more rapid epiphyseal maturation. These observations allow us to infer the dose-response relationship between circulating estradiol levels and the growth rate and bone maturation of children and adolescents. Estradiol levels observed in prepubertal boys (0.08pg/ml (0.3 pmol/1)) appear to be without effect on growth rate or bone maturation. We hypothesize that estradiol levels in prepubertal girls (0.6pg/ml (2.2 pmol/1)) cause the 20% more rapid epiphyseal maturation that is observed in girls compared to boys. T h e absence of this estradiol in girls with Turner's syndrome may explain their corresponding delay in bone maturation compared to normal girls [15]. Still higher levels of estradiol begin to exert major effects on growth velocity, with the maximal growth rate during male puberty corresponding to estradiol levels of approximately 3 - 4 pg/ml (1115 pmol/1) [16]. Further increases in estradiol level, to 12 pg/ml (44 pmol/1) and above, progressively appear
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to uncouple growth velocity from epiphyseal maturation and fusion. In conclusion, pubertal growth and skeletal maturation in both sexes is driven primarily by estrogen. T h e dose-response curve for the growth-promoting action of estrogen is biphasic, with maximal stimulation of growth at estradiol levels of approximately 3 - 4 pg/ml (11-15 pmol/1). Androgen at or above normal adult male levels does not fuse the epiphyses. Rather, it is estrogen that fuses the epiphyses and terminates linear growth. Lastly, prepubertal girls have eight-fold higher estradiol levels than prepubertal boys, which may explain their more rapid epiphyseal maturation.
REFERENCES 1. Cutler G. B. Jr, Cassorla F. C., Ross J. L., Pescovitz O. H., Barnes K. M., Comite F., Feuillan P. P., Laue L., Foster C. M., Kenigsberg D., Caruso-Nicoletti M., Garcia H. B., Urairte M. M., Hench K. D., Skerda M. C., Long L. M. and Loriaux D. L.: Pubertal growth: physiology and pathophysiology. Recent Progr. Horm. Res. 42 (1986) 443-470. 2. Ross J. L., Cassorla F. G., Skerda M. C., Valk I. M., Loriaux D. L. and Cutler G. B. Jr: A preliminary study of the effect of estrogen dose on growth in Turner's syndrome. N. Engl. J. Med. 309 (1983) 1104-1106. 3. Ross J. L., Long L. M., Skerda M. C., Cassorla F. G., Loriaux D. L. and Cufler G. B. Jr: Effect of low doses of estradiol on six month growth rates and predicted height in patients with Turner syndrome. J. Pediatr. 109 (1986) 950-953. 4. Ross J. L., McCauley E., Roeltgen D., Long L., Kushner H., FeuiUan P. and Cutler G. B. Jr: Self-concept and behavior in adolescent girls with Turner syndrome: potential estrogen effects. J. Clin. Endocr. Metab. 81 (1996) 926-931. 5. Caruso-Nicoletti M., Cassorla F. G., Skerda M. C., Ross J. L., Loriaux D. L. and Cutler G. B. Jr: Short term, low dose estradiol accelerates ulnar growth in boys. J. Clin. Endocr. Metab. 61 (1985) 896-898. 6. Shenker A., Laue L., Kosugi S., Merendino J. J. Jr, Minegishi T. and Cutler G. B. Jr: A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365 (1993) 652-654. 7. Laue L., Chan W. Y., Hsueh A., Wu S. M., Blomberg L. and Cutler G. B. Jr: Genetic heterogeneity of constitutively activating mutations of the luteinizing hormone receptor in familial male-limited precocious puberty. Proc. Nam. Acad. Sci. U.S.A. 92 (1995) 1906-1910. 8. Laue L., Kenigsberg D., Pescovitz O., Hench K. D., Barnes K. M., Loriaux D. L. and Cutler G. B. Jr: Treatment of familial male precocious puberty with spironolactone and testolactone. N. Engl. J. Med. 320 (1989) 496-502. 9. Smith E. P., Boyd J., Frank G. R., Takahashi H., Cohen R. M., Specker B., Williams T. C., Lubahn D. B. and Korach K. S.: Estrogen resistance caused by a mutation in the estrogenreceptor gene in a man. N. Engl. J. Med. 331 (1994) 10561061. 10. Federman D. D.: Life without estrogen. N. Engl. J. Med. 331 (1994) 1088-1089. 11. Morishima A., Grumbach M. M., Simpson E. R., Fisher C. and Qin K.: Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J. Clin. Endocr. Metab. 80 (1995) 36893698. 12. Greulich W. W. and Pyle S. I.: Radiographic Atlas of Skeletal Development of the Hand and Wrist. Stanford University Press, Stanford, CA, 1959.
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15. Midtbo M. and Halse A.: Skeletal maturity, dental maturity, and eruption in young patients with Turner syndrome. Acta OdontoL Scand. 50 (1992) 303-312. 16. Klein K. O., Martha P. M. Jr, Blizzard R. M., Herbst T. and Rogol A. D.: A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. II. Estrogen levels as determined by an ultrasensitive bioassay..7. Clin. Endocrinol. Metab. 81 (1996) 3203-3207.