The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 12 4677-4694
Copyright © 1999 by The Endocrine Society
Estrogen: Consequences and Implications of Human Mutations in Synthesis and Action1
Melvin M. Grumbach and
Richard J. Auchus
Department of Pediatrics, School of Medicine, University of
California–San Francisco, San Francisco, California
94143-0434
Address correspondence and requests for reprints to: M. M. Grumbach, Department of Pediatrics, School of Medicine, University of California–San Francisco, San Francisco, California 94143-0434. E-mail: grumbac{at}itsa.ucsf.edu
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Abstract
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Recent developments have advanced our knowledge of the role of estrogen
in the male. Studies of the mutations in CYP19, the gene encoding
aromatase, in six females and two males and a mutant estrogen receptor
in a man are described. These observations provide illuminating new
insights into the critical role of estrogen in the male (as well as
female) in the pubertal growth spurt and skeletal maturation, and in
the importance of estrogen sufficiency in the accrual and maintenance
of bone mass. The weight of evidence supports an effect of androgens on
the latter processes, but this effect has not been quantitated.
There is a discordance in the estrogen-deficient male between skeletal
growth and skeletal maturation and the accrual of bone mass and
density. Estrogen synthesis by the testis is limited before puberty,
and estrogen deficiency does not affect the age of pubertal onset.
Estrogen deficiency in men leads to hypergonadotropism, macroorchidism,
and increased testosterone levels. Estrogen lack has a significant
effect on carbohydrate and lipid metabolism, and estrogen resistance
was associated with evidence of premature coronary atherosclerosis in a
man. These observations have highlighted the role of extraglandular
estrogen synthesis and intracrine and paracrine actions.
In the human, in contrast to nonprimate vertebrates, aromatase
deficiency and estrogen resistance () does not seem to affect gender
identity or psychosexual development. The clinical repercussions of
mutations in CYP19 on the fetal-placental unit have highlighted the
major role of placental aromatase in the protection of the female fetus
from androgen excess, thus preventing androgen-induced
pseudohermaphrodism and virilization of the mother. These features are
compared with the virilization that occurs in utero in
the female spotted hyena.
The novel features of the aromatase deficiency syndrome in the affected
female—in the fetus, during childhood, and at puberty—are discussed,
including virilization at puberty and development of polycystic
ovaries. The severity of the syndrome correlates with the severity of
impairment of aromatase formation in expression systems.
Finally, the structural consequences of missense mutations in CYP19 are
described in accordance with a model of the structure of human
aromatase.
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Introduction
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LONG HELD concepts of the role and effects
of estrogen in the male have been challenged by recent discoveries.
Historically, the role of estrogen in the development, growth, and
function of the human male has been thought to be relatively
unimportant and minor. A remarkable change in perspective has recently
emerged. Until 1996, only one estrogen receptor (cloned in the
mid-1980s) (1, 2) was thought to mediate the effects of estrogen.
Furthermore, even though in the human the enzyme (encoded by the CYP19
gene) P450 aromatase—the critical enzyme responsible for the last and
irreversible step in estrogen synthesis from androgens—was recognized
to be expressed in multiple tissues in the 1990s (3, 4, 5), the
significance of estrogen in male physiology had not been
appreciated.
New developments have challenged traditional constructs and advanced
our understanding of the widespread effects of estrogen in diverse
functions in the male, stimulating research ranging from integrative
physiology to cell and molecular biology within and outside the
reproductive system. Three developments are largely responsible for
these advances: 1) description of a man with a null mutation in the
ER that caused estrogen unresponsiveness (6) and of men and girls
and women (7, 8, 9) with severe estrogen deficiency due to autosomal
recessive mutations in the gene encoding aromatase; 2) the concurrent
development of mice that lack the estrogen receptor (ERKO mice)
(10, 11, 12, 13) or the gene encoding aromatase (ArKO mice) (14); and 3) the
discovery of a second widely distributed estrogen receptor, ERß
(15, 16, 17, 18, 19, 20, 21), and, most recently, generation of the estrogen receptor ß
knockout mouse (ßERKO) (22) (Fig. 1
).
In addition to the relatively slow action of estrogen through classic
intracellular steroid receptors (ligand-regulated transcriptional
factors that interact with so-called transcriptional coregulatory
proteins—coactivators and corepressors to stimulate or inhibit gene
expression in target tissues) (23, 24), estrogen also acts through
nongenomic processes involving steroid receptors on the cell surface to
mediate rapid effects of the hormone on certain cells and tissues
(25, 26, 27, 28, 29).
Unlike genes for many steroidogenic enzymes, such as CYP17 and CYP21,
the large gene (>75 kb) encoding aromatase is expressed in many human
tissues (in contrast to the situation in nonprimate vertebrates). The
tissue-specific expression of the enzyme is regulated by means of
tissue-specific promoters, but the translated protein is the same in
all tissues (see Refs. 3, 4, 5) (Fig. 2).
These tissues include the placenta and preimplantation blastocyst, the
brain, ovary and testis, adipose tissue, fetal liver, muscle, hair
follicles, bone, pituitary gland, and the immune system.
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Figure 2. The structure of the large human CYP19 gene
(P450arom) and the mutations that cause aromatase deficiency. The
numbered black boxes designate translated exons. The
septum in the open box in exon II represents the 3' acceptor splice
junction for the untranslated exons. The multiple alternate promoters
and the untranslated exons are designated by open boxes. The promoter
I.1, which lies more than 40 kb 5' of the translation start site, is
responsible for expression of aromatase in the placenta; promoter I.4
is predominant in adipose tissue and the proximal promoter II is the
major promoter in the gonads. The distinct P450 arom promoters provide
tissue specific expression by alternate splicing with the common 3'
acceptor splice junction in exon II; irrespective of the choice of the
various promoters, only a single aromatase protein is expressed. The
known mutations are shown. X indicates a nonsense (stop)
mutation. Three mutations are in the heme binding region (HBR). GT 
GC + 29aa is a thymidine to cytosine transition at the splice junction
between exon VI and intron VI, giving rise to a 29 amino acid insert in
aromatase. From Grumbach MM, Conte FA. Disorders of sex
differentiation. In: Wilson JD, Foster DW, Kronenberg HM, Larsen PR,
eds. Williams textbook of endocrinology, 9th ed. Philadelphia: W.B.
Saunders Co. Ltd., 1998; 1303–1425. Modified from Ref. 9.
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Over 25 years ago, Siiteri and MacDonald (30) and Tait and his
associates first quantified the extragonadal, extra-adrenal synthesis
of estrogen in pregnant and nonpregnant women, in men, and in men and
women with a variety of clinical disorders. New developments have
highlighted the complexity of extraglandular estrogen synthesis from
androgens and androgen precursors (mainly androstenedione,
testosterone, and dehydroepiandrosterone and its sulfate) by the
aromatase enzyme. These advances focus attention on intracrine
mechanisms synthesis (i.e. the tissue- and
substrate-specific synthesis in peripheral cells from circulating
steroid precursors of androgens and estrogens, which act within the
specific cell and are not released) and the paracrine and autocrine
roles of locally produced estrogen (31, 32, 33). A wide variety of cells,
for example, contain both aromatase and estrogen receptors (Fig. 3).
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Figure 3. The complexity of extra-glandular synthesis
of estrogen hormones by the conversion of C19 androgens or androgen
precursors to aromatic C18 estrogens diagrammatically represented.
Intracrine mechanisms (see text) refer, in this instance, to the
synthesis in peripheral cells of estradiol from testosterone and other
19-carbon precursors. Testosterone (T) entering the cell from the
circulation is converted to dihydrotestosterone (DHT) by 5R (5
reductase 1 or 2), which acts through binding to the androgen receptor
(AR). T is converted to E2 (17ß-estradiol) by CYP19, and
the E2 binds to either the ER or ERß forming homo- or
heterodimers. Intracellular synthesized T, for example, can arise from
 4A (androstenedione) and DHEA or DHEAS. The
desulfated DHEA is converted to 4A by
3ß-hydroxysteroid dehydrogenase and the 4A is
transformed into T by 17ß-hydroxysteroid dehydrogenase
(e.g. Type V isoenzyme), which then can be converted to
E2; or androstenedione can be converted to estrone and then
to E2 by the Type I isoenzyme. Some of the family of
17ß-hydroxysteroid dehydrogenase isoenzymes (e.g. Type
II isoenzyme) can convert E2 to estrone, providing an
additional mechanism of the regulation of estrogen synthesis and
metabolism. The E2 synthesized by intracrine mechanisms can
be released to act on a neighboring cell—a paracrine
mechanism—through the cell’s estrogen receptors; for example,
estradiol synthesized by a mesenchymal cell acting on a neighboring
epithelial cell. The E2 also can enter the circulation, an
endocrine role. For comparison, an autocrine mechanism is illustrated.
IGF-I generated in and released by a peripheral cell can act on the
same cell through the cell’s surface IGF-I receptors. Recent studies
have emphasized the importance of the autocrine/paracrine role of IGF-I
in body growth in contrast to the endocrine role of IGF-I synthesized
and released into the circulation by the liver, the major contributor
to plasma IGF-I. Mice with a selectively and totally deleted hepatic
IGF-I gene have greatly reduced circulating IGF-I, but normal postnatal
bone and body growth; these observations challenge the widely held
somatomedin hypothesis (198 199 ). Similarly, even though estrogens
produced by extraglandular synthesis are a major source of circulating
estrogen in the male and the postmenopausal woman, especially, the
intracrine and paracrine role of estrogen in its diverse and
specialized functions in specific tissues needs to be considered.
Endocrine, paracrine, and intracrine estrogen can also act rapidly on
cell surface receptors, a nongenomic action.
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In addition, the exceptional tissue distribution and differential
actions of ER and -ß receptors and their multiple isoforms
(34, 35, 36) and their capacity to form heterodimers as well as homodimers
(37), combined with the interplay of androgen and estrogen action on
target tissues (38, 39, 40), provide a new conceptual framework for
estrogen action. These advances, coupled with the development of
synthetic, tissue-selective nonsteroidal and steroidal estrogen
receptor agonists and antagonists–selective estrogen receptor
modulators (SERMs)—have important therapeutic applications (24, 41, 42). Table 1 lists the diverse sites of
action of estrogen.
The present discussion focuses on the clinical repercussions of human
mutations that lead to estrogen resistance or deficiency in the human
and have provided insight into the role of estrogen in the male and the
role of the placenta in protecting the female fetus and the mother from
virilization. The discovery of a young adult man with a null mutation
of ER clarified the role of estrogen acting through the ER
receptor on bone. The description of two males and six females with
estrogen deficiency due to mutations in CYP19 (Fig. 2
) has shown the
major role of aromatase in the fetal placental unit, protecting the
female fetus and mother from androgen excess; the critical role of
estrogen in pubertal maturation, in bone accrual and growth, and in
skeletal maturation and epiphyseal fusion in both the male as well as
the female; and have clarified the action of estrogen in carbohydrate
and lipid metabolism. Neither estrogen resistance or deficiency seems
to affect gender identity.
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Estrogen and Its Effects on Pubertal Growth, Skeletal Maturation,
and Bone Accrual
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Estrogen has manifold effects on bone including a critical, but
incompletely understood, action in the pubertal growth spurt, in
skeletal maturation, and in the arrest of linear growth by fusion of
the epiphyseal growth plate, in the accrual of peak bone mass, and in
its maintenance and repair. These dynamic actions (including
interactions with the GH and insulin-like growth factors (IGF) and
their binding proteins, other growth and morphogenic factors, thyroid
hormone, vitamin D, retinoids, PTH and PTHRP, cytokines, and
their receptors among many factors) involve a variety of complex
developmental and homeostatic programs (see Ref. 43).
The mechanisms involved in the epiphyseal fusion of long bones–the
transformation of the cartilaginous growth plate into bone–is mediated
in large part by the action of estrogen during puberty. This action
includes a program of orderly proliferation, maturation, apoptosis of
chondrocytes, proteolysis of the cartilage extracellular matrix, and
vascular and osteoblast invasion of the growth plate. Among other
factors (44), gelatinase B (45) and vascular endothelial growth factor
(VEGF) (46) are essential signals in the ossification process. It is
not known whether estrogen has an effect on these important factors in
osteogenesis. However, in the mammary gland of the baboon, estradiol
stimulated VEGF messenger RNA (47) and estradiol increases VEGF
messenger RNA and VEGF protein in the human MCF-7 breast carcinoma line
(48). Stimulation by estrogen of VEGF in endothelial cells in bone
could provide one explanation for its capacity to increase the rate of
epiphyseal fusion. Estradiol-17ß (but not its inactive isomer) also
increases protein kinase C activity in chondrocytes from the rat growth
plate, apparently involving a nongenomic action (49).
The constancy of bone mass in maturity is maintained by bone
remodeling—the critical balance of bone formation and resorption.
Resorption of bone by osteoclasts leads to bone formation by mature
osteoblasts, the principal effector cells. Although closely linked,
these processes are not necessarily regulated by each other, but can
occur independently (50). Estrogen (and androgen) affects bone
formation, an anabolic effect, and has a well characterized
antiresorptive action that involves its action on local cytokine
production (51, 52, 53).
In males, constitutional delay in onset of puberty has been advanced as
a cause of decreased peak bone mass (54, 55); however, a recent study
has disputed this contention (56). On the other hand, sexual
infantilism due to hypergonadotropic or hypogonadotropic hypogonadism
is associated with decreased peak bone mass. Hypogonadism can also lead
to osteoporosis if untreated with estrogen in the female or
testosterone in the male; rapid bone loss ensues within 5 yr of
castration or the onset of severe hypogonadism. In a long-term study of
72 hypogonadal males, which included serial determinations of
volumetric bone mineral density (vBMD) of the lumbar spine, continuous
testosterone replacement therapy returned the vBMD into the normal
range (57). What mediates this anabolic and antiresorptive effect of
testosterone on bone?
Estrogen had been recognized as the major sex steroid in the female,
responsible for the pubertal growth spurt, skeletal maturation, and the
accrual of bone mass (although some held the view that androgens and
androgen precursors secreted by the ovary and adrenal were an important
factor in these processes); these effects of estrogen were not thought
to be important in the male. At puberty in the female, the increased
synthesis and secretion of estrogen by the ovary causes the progressive
skeletal maturation that eventually leads to epiphyseal fusion and the
termination of linear growth. In the male, on the other hand, received
wisdom dictated that testosterone, the corresponding male sex steroid,
secreted by the testis was directly responsible for these events during
puberty (58) with estrogen having a minor effect, if any.
The reports of a 28-yr-old sexually mature male with tall stature,
unfused epiphyses, osteopenia, eunuchoid skeletal proportions, and
progressive genu valgum (6) due to an autosomal recessive inherited
mutation in the ER (Table 2) and of
two adult males with severe estrogen deficiency due to autosomal
recessive transmitted mutations in the gene encoding aromatase (9, 59, 60, 61) (Table 2) have changed our thinking. All three men had a normal
age of onset of puberty and identical clinical findings despite high or
normal testosterone concentrations (Table 3), documenting the critical role in the
male of estrogen in skeletal maturation, the accumulation of bone mass,
and the pubertal growth spurt. In the two men with aromatase
deficiency, but not in the man with estrogen resistance, treatment with
estrogen led to rapid skeletal maturation and epiphyseal fusion at the
wrist within 6–9 months (59, 60) and a dramatic increase in bone
mineralization (61). Fig. 4 shows the
growth pattern and bone age and the effect of estrogen treatment in
this patient (61). Fig. 5 presents the
effect of estrogen treatment on the accrual of bone mass (61).
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Figure 4. The linear growth curve (•) and bone age
(panels) of the 24-yr-old man with aromatase deficiency whose height
was 204.5 cm (+3.7 SD). Note the continued steady growth
rate without an apparent pubertal growth spurt, which led to very tall
stature and rapid cessation of growth after the institution of estrogen
therapy (bar). The X denotes a bone age for the chronological age
(dashed line). The open epiphyses at the wrist
(left) closed within 6 months of treatment
(right). The dark curve is the average normal value for
age, and the numerals indicate SD from the mean value (9 61 ). The growth curve and, of interest, height and bone age (data not
shown), were almost identical in the man with a nonsense mutation in
the estrogen receptor protein (61 ). From Bilezikian JP, Morishima
A, Bell J, Grumbach MM. 1998 Increased bone mass as a result of
estrogen therapy in a man with aromatase deficiency. N Engl J Med
339:599–603.
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Figure 5. The dramatic change in bone density during
estrogen therapy in the man with CYP19 deficiency. The initial daily
dose of 0.3 mg of conjugated estrogens was gradually increased during
the first 12 months to 0.75 mg daily, his present dose (see text). On
this dosage, the accrual of bone density continued after growth ceased
(within 6 months) and increased to within the normal range for adult
young men without inducing gynecomastia, impotence, or excessive weight
gain. Following baseline studies, bone density was measured at 12, 30,
and 36 months (vertical lines) (61 ). Modified from Bilezikian JP,
Morishima A, Bell J, Grumbach MM. 1998 Increased bone mass as a result
of estrogen therapy in a man with aromatase deficiency. N Engl J Med
339:599–603.
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These three very remarkable men, together with observations in and
ß ERKO and ArKO mice, have led to a revolution in thought about the
importance of estrogen in the male, and the recognition of important
species differences (see below).
Pubertal growth spurt
Despite normal or increased (the aromatase-deficient men) plasma
concentrations of testosterone, none of the men had an apparent
pubertal growth spurt (Fig. 4). Whereas the evidence provided by
examination of their growth curves is not definitive because of gaps in
measurement, the data suggest continued steady, slow growth throughout
adolescence and the 3rd decade without a "pubertal inflection" or
epiphyseal fusion. The delay in skeletal maturation with continued
growth leads to eunuchoid skeletal proportions (9). These observations
strongly support an essential role for estrogen in the pubertal growth
spurt in the male.
Not only does a rise in estradiol concentration correlate with the
earlier onset of the pubertal growth spurt in normal girls, but in boys
sampled longitudinally before and during puberty, the rise in the
estradiol levels as measured by an ultrasensitive bioassay correlated
with peak height velocity which occurred, as expected, about 3 yr after
pubertal onset (62). Of note, the plasma estradiol concentrations
during peak height velocity are similar in boys and girls (about 3–4
pg/mL) and correlate not only with testosterone levels in boys, but
with bone age as well.
Additional critical support is provided by the pattern of growth in
46,XY phenotypic females with the complete androgen insensitivity or
resistance (CAIS) syndrome. These women have a pubertal growth spurt
despite resistance to the action of testosterone owing to a mutation or
deletion of the androgen receptor. In the study by Zachmann et
al. (63), the adult height of CAIS patients was 172.3 ± 4.1
cm SD, 10 cm greater than normal women
(162.2 ± 6.0 cm SD), but 2.4 cm less than
normal men (174.7 ± 6.7 cm SD). The greater
mean height of CAIS than normal women may be related in these 46,XY
individuals, in part, to Y-specific growth genes outside of the
pseudoautosomal region on the short arm of the Y chromosome. The age of
peak height velocity and the pattern of skeletal maturation and
epiphyseal fusion were comparable to normal females. Zachmann et
al. (63) advanced these observations as evidence for the
importance of estrogen alone in the female pubertal growth, as opposed
to the role of ovarian or adrenal androgens. This study indicated that
despite high testosterone concentrations in the presence of
physiologically ineffective testosterone action, the pubertal growth in
these XY women is attributable to estrogen secreted by the testis and
arising from extragonadal conversion of androgens to estrogens (the
latter occurs independent of the androgen receptor). Similarly, for
example, the autosomal dominant aromatase excess syndrome in
prepubertal age boys, in addition to gynecomastia, is associated with
an increased rate of growth and skeletal maturation despite a
prepubertal concentration of plasma testosterone (64, 65, 66, 67). Comparable
observations have been reported in boys below age 6 with the
Peutz-Jeghers syndrome and estrogen-secreting testicular tumors
(68, 69, 70).
Other subtle and suggestive, but not definitive, clues to the effect of
estrogen on growth and skeletal maturation in the male are listed in
Table 4. The low plasma concentrations of
estradiol during peak height velocity suggest a potent effect of
circulating estradiol on linear growth, but at least two other factors
need to be considered in the process: the local aromatization of
androgen in bone and the effect with pubertal onset of increased GH
secretion and IGF-I generation (reviewed in Refs. 70, 71). Even
though GH secretion increases 2- to 3-fold during puberty, a normal
pubertal growth spurt can occur without increasing the dose (per kg) of
rhGH therapy in children with severe GH deficiency (58, 72).
In summary, estrogen, not testosterone, has a critical role in the male
pubertal growth spurt and in ensuring normal skeletal proportions as
exemplified by males with estrogen resistance or deficiency.
Bone maturation and the accrual of bone mass during puberty
Aromatase is expressed widely in human and rodent bone, including
in osteoblasts (73, 74, 75, 76, 77, 78, 79), chondrocytes of articular cartilage and bone
adipocytes (73). Both estrogen receptors, and ß, are expressed in
human bone; ER is expressed in osteoblasts (80, 81, 82) and in human
osteoclasts (52) (although the latter remains controversial; Refs. 82, 83), and ERß is expressed in osteoblasts, but less so than ER
(84). ER is expressed in resting, proliferative, and hypertrophic
human growth plate chondrocytes (82); in contrast, ERß was identified
only in hypertrophic epiphyseal chondrocytes (41, 85).
With the onset of puberty there is a rapid increase in bone mass
(86, 87, 88, 89, 90, 91), which correlates with bone age. The rate of bone mass accrual
approaches a peak in girls by 16 yr, about 3 yr after menarche, and in
boys by about 17 yr; the rate then decreases, reaching a plateau in the
3rd decade. For example, white girls attained 94% of volumetric bone
density by age 17 yr; in contrast, white boys attained only 86% of
volumetric bone density by 17 yr of age (90). Bone acquisition during
childhood and through puberty is a major determinant of peak bone mass,
which is influenced by genetic and a variety of other factors, as well
as hormones. In both sexes, this peak occurs after peak height
velocity; hence, the attainment of peak bone mass lags behind the
increase in linear growth.
The men with estrogen deficiency or estrogen resistance have severe
osteopenia as quantified by dual-energy x-ray absorptiometry scans and
an increased rate of bone turnover, as evidenced by the markers of
osteoblastic and osteoclastic activity (Table 5). Treatment with high doses of estrogen
had no effect on growth, the skeleton, or classic estrogen end organs
in the man with the null mutation of the estrogen receptor (6). In
contrast, a relatively low dose of conjugated estrogen (0.3 mg/day
gradually increased to 0.75 mg/day over 12 months) had a dramatic
positive effect on biological markers of bone metabolism, bone mass and
maturation, as well as other sites of estrogen action (see below),
without evoking gynecomastia (59, 61). Epiphyseal fusion at the wrist
was rapid in both men with severe aromatase deficiency (59, 60, 61). In the
patient described by Morishima et al. (9) (who had fused
proximal femoral epiphyses but not ossification of the iliac
apophyses), epiphyseal fusion occurred by the 6th month of estrogen
treatment and, as a consequence, linear growth ceased (Fig. 4), but
bone mineral accrual continued (Fig. 5) (61). Within 3 years of
initiating estrogen therapy, the markers of bone turnover gradually
approached normal values (Table 6). The
excretion of urinary calcium fell, and bone mass increased
dramatically. Bone mass in the lumbar spine had increased by 20.7%, in
the femoral neck by 15.7%, and in the distal radius by 12.9% (61). In
this estrogen-treated aromatase-deficient man, all but the radial site
was at the mean value for normal young men (Fig. 5).
In the young male adults with aromatase deficiency, estrogen treatment
not only prevented increased bone loss but repaired the severe
osteoporosis—a striking anabolic effect on bone mineral content and
volumetric and areal BMD mediated, at least in part, by stimulation of
osteoblastic synthesis of IGF-I and transforming growth factor-ß (61, 92). In menopausal women, on the other hand, exogenous estrogen in the
traditional dosage range primarily protects against bone loss (61, 93).
This difference in estrogen action, an anabolic in contrast to a
primarily protective (antiresorptive) effect, in part, may be an
age-related (94) or dose-related phenomenon (95). The osteoporotic bone
of postmenopausal women apparently has a limited capacity to mount an
anabolic response to conventional estrogen replacement (94), unlike
that of the young adult.
The two girls with aromatase deficiency (one a sibling of the affected
man) we studied during adolescence had a delayed bone age despite
increased circulating androgens and virilization at puberty (8, 9).
Estrogen therapy induced a pubertal growth spurt and epiphyseal fusion
in both girls. A female child with a null mutation in the P450 arom
gene (CYP19) had densitometric evidence of osteopenia of the lumber
spine at age 3 4/12 yr that improved after 50 days of low-dose estrogen
therapy (96).
Thus, in both males and females the lack of estrogen leads to a
dissociation between skeletal growth and skeletal maturation and the
accrual of bone density and mass.
Prepubertal estrogens may be important in the sex difference in the
rate of skeletal maturation. A bone age of 13 yr in boys is equivalent
in the standard gender-specific estimates of skeletal age to that of an
11-yr-old girl; the bone maturation of girls in childhood is about 20%
more advanced than that of boys. The nature of this difference had not
been understood. Whereas RIA were sufficiently sensitive and specific
to quantitate the low testosterone levels in prepubertal boys (97, 98),
the method of estimating low (pg/mL) amounts of serum estradiol (99),
despite many reports to the contrary, was not sufficiently sensitive
and specific to detect prepubertal levels in girls, much less in boys
even after solvent extraction and thin-layer chromatography (100).
Although a prepubertal sex difference in plasma estradiol
concentrations was suspected, the hypothesis remained speculative until
Klein et al. (101) developed an ultrasensitive recombinant
cell bioassay for serum estradiol (detection limit 0.02 pg/mL). With
this assay, prepubertal girls (age 7.5 ± 2.1 yr
SD) had a mean concentration of serum estradiol
of 0.6 ± 0.6 pg/mL, whereas the level in prepubertal boys was
0.08 ± 0.2 pg/mL; no correlation with age or body mass index was
found. This more than 7-fold difference was proposed to explicate the
more advanced skeletal maturation of prepubertal girls compared with
boys (101). Moreover, aromatase activity was not detected or present at
a low level in the Leydig cells of prepubertal boys (102, 103), nor was
aromatase detected in the human fetal testis at 16–18 weeks of
gestation (69). Aromatase is not well expressed in the testis until LH
rises at the time of male puberty (102, 103, 104). Furthermore, the total
aromatase activity of adipose tissue and other extraglandular tissues
is low in childhood (64, 105). Accordingly, in the male, circulating
estrogen and intracrine and paracrine estrogen becomes important in
growth and skeletal maturation during puberty, but not at its
onset.
These observations indicate that estrogen has an essential role in the
pubertal growth spurt, skeletal maturation, and the accrual of a normal
peak bone mass in males as well as females and may have an effect on
the prepubertal accretion of bone mineral in the female. New
therapeutic approaches—the use of nonsteroidal third generation
aromatase inhibitors (e.g. letrozole,
anastrozole) and ER antagonists, including SERM—to control growth
and skeletal maturation in disorders of growth and puberty by
suppression of estrogen synthesis or action are suggested by the
critical role of estrogen in these processes (Table 7). The use of these pharmacological
agents in the male should not delay or affect the appearance of male
secondary sex characteristics. Furthermore, they suggest that in the
human ER, but not ERß, is the principal estrogen receptor that
mediates the action of estrogen on bone mass accrual and epiphyseal
fusion. Additional support is indicated by the bone findings, still
preliminary, in the ERKO (11, 106) and ArKO mouse (Simpson, E.R.,
personal communication), but there are important species differences.
Table 8 summarizes the effect of estrogen
in the male and female on pubertal growth and bone.
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Table 7. Potential use of aromatase inhibitors (or estrogen
receptor antagonists) in disorders of growth and sexual maturation to
restrain skeletal maturation
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Androgens, Growth, and Bone Mass Accrual
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Androgens can act directly by activating the androgen receptor or
indirectly by their conversion by aromatase to estrogen. The
observations in the estrogen-deficient and -resistant men have
redefined the role of androgens in the skeleton. Despite normal or
strikingly elevated testosterone concentration in the men with impaired
estrogen synthesis or action, a pubertal growth spurt was not apparent,
and a steady rate of growth that continued into adulthood was
associated with lack of epiphyseal fusion, tall stature, eunuchoid
skeletal proportions, and marked osteopenia. Rapid fusion of the
epiphyses with the initiation of estrogen therapy in the
estrogen-deficient men (59, 60, 61) was accompanied by the gradual
accretion of a normal bone mineral content (61).
It is important to emphasize that establishing a role for estrogen does
not exclude a direct effect of androgen on bone in the male (but one
less important than traditionally thought). Quite likely, the direct
effect of testosterone (not mediated by conversion to estradiol) is
small with reference to pubertal growth and epiphyseal fusion. However,
the effect of estrogen treatment on bone mass accrual in the aromatase
deficient males occurred in the presence of normal or increased
testosterone concentrations. A significant body of evidence supports a
direct role for androgen on bone mass and in the sexual dimorphic
features of the skeleton (107, 108, 109, 110, 111). The human androgen receptor is
present in a variety of bone cells, including osteoblasts (112),
osteoclasts (108, 113), osteocytes (114), and hypertrophic growth plate
chondrocytes (114). The androgen receptor is expressed in periosteal
bone (114) as well as cancellous bone. That androgens have a
significant role in bone mass accrual is supported by evidence reviewed
by Hofbauer and Khosla, (111) Orwoll (107), and Kasperk et
al. (109) in the human and, for example, by Vanderschueren
et al. (115, 116, 117, 118, 119) and Lea and Flanagan (120) in the rat and
by Maor et al. (121) in the mouse. In many studies in the
rodent, aromatizable androgens (e.g. testosterone,
androstenedione) were used and not dihydrotesterone or synthetic
androgens that are not substrates for aromatase, and they confound
interpretation of the direct action of androgen on bone (111). However,
5-dihydrotestosterone and some synthetic androgens can increase the
rate of growth, and, synthetic androgens, the rate of skeletal
maturation of children, without increasing circulating GH or IGF-I
concentrations.
Furthermore, the decline in bone mass in elderly men correlates better
with free estradiol concentrations than with free testosterone levels
(122, 123). Men have greater cortical bone width and, as would be
predicted from their taller size and higher bone mass, testosterone
probably stimulates the periosteal growth of cortical bone (124). Of
interest, women with ovarian hyperandrogenism have high BMD (125).
Moreover, estrogen treatment of male to female transsexuals increases
BMD and markers of bone remodeling (126, 127).
As a counterpart to the action of estrogen in the male on pubertal
growth and the skeleton, one can turn to the CAIS in the 46,XY
phenotypic female who is unresponsive to androgens because of a
deletion or null mutation in the X-linked gene encoding the androgen
receptor. In these women, the testes secrete estradiol (99) and
testosterone, and aromatase activity in the testis and in peripheral
tissues is intact. As mentioned earlier, these women have a pubertal
growth spurt and fuse their epiphyses in the absence of androgen
action. The effect of this mutation on bone mass accrual and bone
turnover is smaller, but many confounding factors, including castration
in childhood or adolescence and the location of the testes, complicate
the interpretation of this phenomenon. Estrogen production, at least in
some individuals with CAIS, although greater than that in the normal
young male adult (99, 128), is less than the estimated mean estrogen
production during the normal menstrual cycle. While both volumetric and
areal bone density are reduced in CAIS, biochemical markers of bone
turnover are normal (129, 130, 131). The skeletal mass in these women is
similar to that of normal women (130). Nor do these patients seem to
have an increased risk of fractures (129). These observations, however,
suggest that the production of estrogen by both the testis and
peripheral tissues is sufficient to induce a pubertal growth spurt,
epiphyseal fusion, and near normal accrual of bone mass for normal
women. Parenthetically the androgen-resistant male rat, analogous to
the human with partial androgen resistance, has reduced bone remodeling
and size, but not decreased bone density (115, 116, 117).
In summary, the conventional belief that the human male skeleton
accrues greater bone mass than that of the female because of the direct
action of testosterone alone seems no longer tenable. Estrogen has an
essential role in attaining optimal peak bone mass in the male.
Nevertheless, androgen seems to have a direct but unquantitated effect
on the skeleton that has not been distinguished unambiguously from the
putative skeletal effects of genes on the human Y chromosome, nor from
the observation that most of the sex differences and the age-related
changes at the end of puberty are attributable to differences in
skeletal dimensions than to bone density.
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The Testis and Ovary and the Regulation of Gonadotropins
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In the man with severe aromatase deficiency whom we studied,
circulating levels of testicular androgens and of FSH and LH were
elevated (9). In the man reported by Carani et al. (60),
whose CYP19 mutation was associated with a lesser degree of aromatase
deficiency, the serum gonadotropins were slightly increased, but the
testosterone level was normal. In the estrogen-resistant man (6),
testosterone concentrations were normal, but estradiol and estrone
levels were more than twice the upper value for the normal range, and
gonadotropin concentrations were increased.
In addition, our patient has macroorchidism (he did not consent to
provide a semen sample before estrogen treatment). The infertile man
reported by Carani et al. (60) had small testes and
oligospermia with immotile on semen analysis, but he had a brother with
a normal CYP19 gene who also was infertile and had azoospermia. The
familial occurrence of infertility in this pedigree limits an
interpretation of the influence of estrogen deficiency on
spermatogenesis. The estrogen-resistant man had normal-sized testes and
a normal sperm count with a sperm viability of 18% (normal >50%).
However, the male ERKO mouse becomes infertile due, at least in
part, to an interruption of fluid resorption by the efferent ductules
of the epididymis, which leads to dilatation and disruption of the
seminiferous tubules (132), whereas the ßERKO male mouse is fertile
but less so than the wild type mouse (22). The male ArKO, initially
fertile, develops progressive infertility associated with impaired
spermatogenesis and a reduction in spermatids (133). In the human male,
in contrast to the rodent, the data are insufficient at present to
allow an assessment of the action of estrogen on spermatogenesis and
fertility.
Treatment with estrogen in the men with aromatase deficiency reduced
the elevated gonadotropin values into the normal range; in our patient
estrogen treatment decreased the high testosterone and
dihydrotestosterone concentrations into the male range and the enlarged
testes reduced to normal (9, 61) (Table 9).
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Table 9. Gonadal hormones, gonadotropins, and testicular size
before and after 3 yr of estrogen therapy in the aromatase-deficient
male
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These observations have clarified further the role of estrogens on the
sex steroid-gonadotropin feedback system in the male (Fig. 6), a site of action for which there was
previous evidence (134, 135). Aromatase and ER predominantly are
expressed in the pituitary (136, 137) and the hypothalamus. In the two
females with aromatase deficiency the elevated levels of androgens
failed to suppress gonadotropins into the normal range after the age of
puberty or in infancy and early childhood in the absence of estrogen
(8, 9), as was the case after puberty in our male patient. In the male,
in the virtual absence of estrogen synthesis, no testosterone,
androstenedione or dehydroepiandrosterone (DHEA) and its
sulfate (DHEAS) were converted to estrogen by the Leydig cells
or extragonadal tissues, and the high LH levels stimulated
hypersecretion of testosterone by the Leydig cells. We attribute the
macro-orchidism in the male patient to the increased level of FSH
acting on a functional testes (9, 61); testes volume decreased after
estrogen treatment. In two female patients, the chronically increased
gonadotropin concentrations led to the formation of multicystic ovaries
at puberty that resolved or were prevented from recurring by
replacement estrogen and progesterone treatment (8, 9). In the affected
female child with a null mutation of the aromatase gene, multicystic
ovaries and hypergonadotropism were present by 2–4 yr of age; low-dose
estrogen therapy led to normalization of gonadotropins and regression
of the ovarian cysts (96).
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Figure 6. The male hypothalamic-pituitary
gonadotropin-testis axis in the light of observations in aromatase
deficiency and defective estrogen receptor. The observations suggest
that estradiol has a significant role in the regulation of FSH and LH.
The plasma estradiol arises from the testes and from aromatization of
19-carbon steroids in extra-glandular tissues; in addition, intracrine
and autocrine/paracrine roles of locally synthesized estradiol in the
pituitary gland and hypothalamus can affect FSH and LH secretion.
Accordingly, estradiol, testosterone, and inhibin all play a part in
the regulation of gonadotropin secretion in males.
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Carbohydrate and Lipid Metabolism and the Cardiovascular
System
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The aromatase-deficient man described by Morishima et
al. (9) had abnormalities of carbohydrate and lipid metabolism
(61). The insulin resistance and the elevated levels of serum
cholesterol, low-density lipoprotein (LDL)-cholesterol triglycerides,
and a low concentration of high-density lipoprotein-cholesterol (Table 10) gradually returned to the normal
range after the administration of low-dose conjugated estrogen
replacement therapy (61) (Table 11).
The man reported by Faustini-Fustini et al. (92) did
not exhibit glucose intolerance but had an abnormal lipid profile (59),
which improved as well with estrogen treatment.
The estrogen receptor -deficient man had axillary acanthosis
nigricans, decreased glucose tolerance, insulin insensitivity, and
increased glycosylated hemoglobin, which did not respond to high-dose
estrogen therapy. This suggests that the insulin resistance was a
consequence, at least in part, of the defective estrogen receptor
or was an independent defect. In contrast to the aromatase-deficient
men, this man had low serum concentrations of cholesterol,
LDL-cholesterol, apolipoprotein (a) and apolipoprotein A1, but normal
serum triglycerides (Table 10). This difference in the serum lipid
pattern from the pattern described in the estrogen-deficient men may be
related, among other factors, to the presence of a functional hepatic
estrogen receptor ß, which may affect lipid metabolism.
The effect of estrogen on carbohydrate metabolism is unclear; estrogen
deficiency is associated with impaired glucose tolerance (138, 139).
However, the normal or elevated testosterone concentration acting in
the absence of estrogen synthesis or estrogen action by the estrogen
receptor may play a role in this phenomenon.
In summary, men with defective estrogen synthesis or action (ERR)
have a propensity for insulin resistance and dyslipidemia.
Estrogen has a cardioprotective effect (140, 141). This seems to
be mediated by two different mechanisms: its effect on serum lipids
(142, 143, 144, 145) and its direct action on human vascular endothelial and
smooth muscle cells and cardiac myocytes and fibroblasts (reviewed in
Refs. 146); both estrogen receptors and ß are present in these
cardiovascular cells and are widely expressed in the cardiovascular
system (147, 148, 149, 150, 151, 152, 153, 154). In addition, there are rapid nongenomic actions of
estrogen on the vascular system (153, 155, 156) and local estrogen
synthesis by aromatase in cardiovascular tissue (157). The man with a
null mutation of the estrogen receptor had an intact rapid (within
5 min) brachial vasodilatory response to sublingual estradiol and an
increase in brachial artery flow velocity (a nongenomic action
involving calcium-activated potassium channels in vascular smooth
muscle cells), but lacked endothelium-dependent vasodilation mediated
by endothelial nitric oxide generation (159), a nongenomic effect on
endothelial cells which may be mediated by a cell membrane estrogen
receptor encoded by the same transcript as the nuclear receptor (29).
By electron-beam computed tomography scan, early coronary artery
calcification indicated the presence of coronary atherosclerosis
despite a low concentration of serum LDL-cholesterol (159) (Table 12). These observations, although
limited to one well-studied patient, are consistent with the importance
of estrogen and estrogen receptor on cardiovascular function and
protection from cardiovascular disease.
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Psychosexual Development and the Central Nervous System (CNS)
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The role of estrogens on psychosexual development in the
human in contrast to other mammals is poorly understood. The men and
women with aromatase deficiency owing to a mutation in CYP19 and the
man with estrogen resistance due to a homozygous mutation in the
estrogen -receptor had sex appropriate gender identities (6, 8, 9).
Taken as a whole, these observations suggest that despite the diffuse
distribution of estrogen receptors and the enzyme aromatase in the pre-
and postnatal CNS (160, 161, 162), and despite well-documented sex
differences in human brain functioning, estrogen in the human does not
have the critical effect on male gender behavior described in
nonprimate mammals (13, 163, 164, 165, 166, 167, 168, 169) and supports restraint in
extrapolating concepts in this active field from studies in animals to
the human (8, 14, 170).
Estrogen receptors and aromatase activity are present in many regions
of the CNS and coexist in some neurons (171), and different effects of
estrogen on neuronal and glial cell growth and function and cerebral
blood flow are mediated by genomic and nongenomic mechanisms (18, 169, 172, 173, 174, 175, 176, 177). For example, estrogen-replacement therapy in postmenopausal
women may have a putative neuroprotective effect (161, 178) and reduce
the risk or delay the onset of Alzheimer’s disease (179) (reviewed in
Ref. 161). Estrogen decreases the generation of Alzheimer ß-amyloid
peptides on neuronal cells in vitro (180) and may improve
short-term memory in postmenopausal women (181).
In women, the menopause and adrenopause reduce the availability of
19-carbon steroid substrates for peripheral conversion to estrogen and,
accordingly, reduce local estrogen formation and action by paracrine,
autocrine, and intracrine mechanisms. In men, 19-carbon precursors
of estrogen, mainly of testicular origin (testosterone and
androstenedione), gradually decline with age but, nevertheless, are
available throughout the aging process for local conversion to estrogen
and may provide a mechanism for a persistent neuroprotective effect of
estrogen on the CNS. Indeed, this analogy extends to bone; there is a
5-fold greater risk of fracture in the subsequent life of 50-yr-old
women as compared with 50-yr-old men.
Table 13 compares and contrasts the
salient features of estrogen receptor -resistance and estrogen
deficiency in the male.
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Mutations in CYP19 in the Female and Placental Aromatase
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A syndrome caused by a variety of autosomal recessive inherited
mutations in the CYP19 gene has been described in six females (Table 14) (8, 9, 96, 154, 182, 184, 185). It
is characterized by androgen-induced female pseudohermaphrodism and
maternal virilization (8, 96, 182), polycystic ovaries, virilization
with lack of female secondary sex characteristics at puberty,
hypergonadotropism, delayed skeletal maturation, tall stature, and
osteopenia (8, 9, 96). The virilization at puberty is associated with
greatly augmented LH-stimulated ovarian androgen synthesis (8, 9).
Estrogen therapy leads to suppression of virilization, regression of
the multicystic ovaries, the development of female secondary sex
characteristics, a pubertal growth spurt, epiphyseal fusion, and repair
of the osteopenia (Table 9) (8, 9). The clinical features are due to
generalized aromatase deficiency.
The lack of placental and fetal hepatic aromatase leads to a failure to
convert fetal adrenal androgen precursors to estrogen (Fig. 7) and results in masculinization of the
external genitalia in the female fetus and, beginning in the second
trimester, virilization of the mother (8, 9, 96, 182), which can be
severe. This provides a dramatic illustration of the critical
importance of placental and fetal hepatic aromatase in protecting the
female fetus and the mother from exposure to large amounts of
testosterone synthesized mainly by the placenta (8, 186).
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Figure 7. The placental synthesis of
testosterone and 18-carbon steroids from 19-carbon precursors in the
CYP19-deficient fetoplacental unit. The placenta lacks the gene
encoding CYP17 and hence is unable to convert 21-carbon steroids to
19-carbon steroids. The black box indicates the
consequences of CYP19 deficiency on the placental synthesis of
estrogens; the enzymatic block leads to the overproduction of
testosterone and androstenedione. 3ß-HSD, 3ß-hydroxysteroid
dehydrogenase 2; 17-ßHSOR, 17ß-hydroxysteroid dehydrogenase 3; DHT,
dihydrotestosterone; 4A, androstenedione;
E1, estrone; E2, estradiol; E3,
estrone. (Reproduced with permission from Conte et al.,
J Clin Endocrinol Metab 1994; 78:1287–1292.)
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At 36–39 weeks gestation, maternal plasma concentrations of estriol,
estrone, and estradiol were strikingly decreased in two mothers with
affected fetuses (96, 182). In marked contrast, maternal levels of
plasma testosterone, androstenedione, dihydrotestosterone, and DHEAS
were increased 4–12 times above the normal mean values (96, 182)
[e.g. the level of plasma-free testosterone was increased
12-fold (96)]. Umbilical cord blood obtained at birth from the two
affected female fetuses had similarly elevated testosterone,
androstenedione, dihydrotestosterone, and DHEAS levels and decreased
estrogen concentrations (96, 182). There is a strong correlation
between the aromatase activity of the mutant CYP19 and the
masculinization of the female fetus and virilization of the mother
(96). As little as 1% of the aromatase activity of wild-type P450arom
(in one allele) protected the mother from severe virilization and was
associated with less masculinization of the genitalia of the female
fetus (Table 15). A pregnant woman with
progressive virilization beginning as early as the second trimester and
who has high circulating androgens and low plasma and urinary estriol
values should be suspected of harboring a fetus (female or male) with a
mutation in the CYP19 gene.
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Table 15. Mutations of CYP19: relation between aromatase
activity in in vitro expression systems and virilization of
the mother
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These observations provide a dramatic illustration of the critical
importance of human placental and fetal hepatic aromatase in protecting
the female fetus and the mother from exposure to excessive amounts of
testosterone of either fetal or maternal origin (8, 186). The
concentration and total amount of placental CYP19 in the
syncytiotrophoblast increases during gestation (Table 16). The mean placental level of
aromatase is 16-fold greater at 40 weeks gestation than at 10 weeks,
and the total aromatase increases 16.5-fold, whereas placental weight
increases 9-fold during this interval of gestation (187). In clinical
states in which this protective mechanism is overloaded with androgens
or androgen precursors either from the fetus or the mother, the female
fetus is at risk for androgen-induced female pseudohermaphrodism (see
Ref. 186). In women carrying a fetus with congenital virilizing adrenal
hyperplasia, for instance, the fetal adrenal enlarges and fetal
androgen precursors increase in the first trimester when placental (and
quite likely hepatic) aromatase is low (187), as manifested by the
relatively low maternal plasma estriol levels at this stage of
gestation (Fig. 8), and exceed at these
levels of substrate the capacity of the fetoplacental unit for
aromatization. The ontogenesis of aromatization during normal gestation
is delicately balanced. The mid-first trimester and early second
trimester is a critical period for male sex differentiation; at this
stage of gestation, the total amount of placental aromatase is
relatively low, and, hence, conversion of androgen to estrogen would
not compromise the level of fetal plasma testosterone concentrations
required for masculinization of the external genitalia. In addition,
estrogen synthesis by the fetus is not required for differentiation of
the Müllerian ducts or female external genitalia (8).
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Figure 8. The pattern of plasma unconjugated estrogens
throughout normal gestation. (Note the log scale for plasma
concentration; gestational age has been calculated from the last
menstrual flow). Estriol is an indirect indicator of the synthesis of
DHEA and 16OH DHEA and their sulfates by the
fetal adrenal cortex and liver and of the capacity for aromatization of
19-carbon steroids by the placenta. Plasma estriol was first detected
at 0.05 ng/mL at 9 weeks gestational age and increased dramatically
over the next 7 weeks (shown by • and thick curve). This is a time in
gestation when the fetal adrenal undergoes rapid growth and when
placental aromatase activity increases. (Modified from Buster JE.
Estrogen metabolism. In: Speroff L, Simpson JL, eds. Reproductive
endocrinology, infertility, and genetics, Vol V of Gynecology and
obstetrics. Hagerstown, MD: Harper and Row, 1980; 1–11.)
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The female spotted hyena is a female pseudohermaphrodite (in contrast
to other members of the hyena family). 
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Abstract
Introduction
