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Massachusetts General Hospital Reproductive Endocrine Science Center Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Stephanie Seminara, Massachusetts General Hospital, Reproductive Endocrine Science Center, Bartlett Hall, Extenstion 5, Boston, Massachusetts 02114.
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Introduction |
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 Top Introduction Inactivating mutations of the...Adrenal hypoplasia congenita and...Kallmann syndrome and the...References |
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Although these principles of GnRH secretion have been established for over two decades, the regulation of GnRH secretion via sex steroid and nonsteroidal factors in normal reproduction (i.e. the menstrual cycle), different stages of the life cycle (i.e. gonadotropin amplification during puberty) and pathophysiologic states (i.e. polycystic ovary syndrome) is a complicated and incompletely understood phenomenon. Patients with single gene mutations within the hypothalamic-pituitary-gonadal axis have provided unique avenues to increase our understanding of reproductive neuroendocrinology. Genetically engineered animal models have further elucidated the roles of specific hormones in reproductive function, by confirming and extending human findings, establishing species-specific phenotypes, or predicting phenotypes when no human model exists.
The combination of both animal and human tools has proven to be powerful, revealing paradoxes of reproductive biology and unexpected differences between species. For example, over 20 yr ago, a naturally occurring mouse model of hypogonadotropic hypogonadism (hpg mouse) was described with low levels of LH and FSH, sexual immaturity, and infertility (6). The hypogonadism was due to a deletional mutation encompassing the distal half of the gene encoding GnRH and the GnRH-associated peptide (GAP) (7); mice homozygous for this mutation had arrested germ cell development but reproductive function could be restored with gene therapy (8). However, despite the obvious candidacy of GnRH as a cause of hereditary hypogonadism in the human, no mutations within the GnRH gene have yet been discovered in this population (9, 10, 11). Therefore, spontaneously occurring mutations in humans and animals as well as targeted disruption of selected genes must be evaluated in a complementary fashion. Examples of some of these mutations, their genotype/phenotype correlations, and the reproductive paradoxes they present, are discussed below.
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Inactivating mutations of the gonadotropin ß subunits and their receptors |
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TopIntroductionInactivating mutations of the...Adrenal hypoplasia congenita and...Kallmann syndrome and the...References |
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-subunit that is shared by both, and the ß- subunit that is
unique and hence confers the specificity to each heterodimer. In both
sexes, LH stimulates steroidogenesis, whereas FSH controls
gametogenesis. In women, LH increases P450c17 expression in thecal
cells and catalyzes conversion of 21-carbon androgen precursors to
androgens that are then aromatized to estrogens in granulosa cells.
Hence, LH is critical for achieving the full complement of ovarian
steroidogenesis, inducing follicular rupture at the midcycle ovulatory
surge, and supporting normal corpus luteum function. In men, LH
stimulates androgen production by Leydig cells, regulating sexual
differentiation, androgenization, sexual function, and fertility. Interestingly, only a single inactivating mutation of the LHß gene has been reported in a young male presenting with delayed puberty and low testosterone and high LH levels that were immunologically active but biologically inactive (12). When challenged with exogenous LH and human CG (hCG), he responded with normal testosterone secretion, demonstrating that Leydig cell function was intact. A homozygous substitution of arginine for glutamine in amino acid 54 was identified in a loop of the ß subunit that was found to inhibit the binding of LH to its cognate receptor. The unique feature of this case was the proband’s normal male external genitalia in the setting of a LH-hormone deficiency. This finding attested to a normal capability for androgen production in utero either from the adrenal gland or from hCG stimulation of testes. Therefore, despite the presence of LH as early as the 10th week of gestation, its role in testosterone stimulation and, by extension, early male sexual development in utero appears to be negligible as opposed to its role in the adult.
While this case demonstrates that LH is not critical for testicular steroidogenesis during fetal development, inactivating LH receptor mutations have been demonstrated to have more severe consequences for male sexual differentiation. Leydig cell hypoplasia (LCH) is a syndrome of abnormal male external genitalia characterized by a lack of any LH/hCG binding and hCG responsiveness in testicular tissue. In 1995, two 46,XY pseudohermaphrodite siblings with LCH (female external genitalia, primary amenorrhea, and lack of breast development) were found to be homozygous for a missense mutation (Ala593Pro) in the sixth transmembrane domain of LH receptor gene (13). Although the mutated receptor bound LH and hCG, no cAMP production resulted from this binding. As more patients with LCH have been genotyped, two clinical subtypes have emerged. Severe LCH is characterized by complete male pseudohermaphroditism with no male secondary sex characteristics, low testosterone in the face of high LH levels, and lack of response to LH/hCG. Less severe disease is characterized by only mild undervirilization with the spectrum of phenotypes related to the specific locations and types of mutations within the LH receptor gene. Interestingly, a 46,XX sibling of the 46,XY pseudohermaphrodite brothers first reported with LCH, and bearing the same mutation, had a relatively mild phenotype—normal secondary sex characteristics, primary amenorrhea, and only low levels of estradiol and progesterone (14). Her ovarian biopsy revealed the presence of all stages of follicular development except for preovulatory follicles or corpora lutea. These histologic findings clearly demonstrate the essential role of LH in follicle rupture and estrogen production.
While the recently reported LH receptor knockout mice represent phenocopies of the human condition, there are some important differences, particularly in the male (15, 16). While humans with severe LH receptor mutations can present as pseudohermaphrodites, knockout male mice have phenotypically normal internal and external genitalia, suggesting a greater role for LH-independent androgen stimulation in this model (15, 16). Spermatogenesis is arrested at the spermatid stage but can be restored with testosterone therapy, although interestingly, the animals remain infertile (16). Female mice more closely resemble the human mutation with preantral and antral follicles present but no preovulatory follicles or corpora lutea, confirming the independence of early follicle development from LH. Taken together, these data suggest that there are different roles for LH in androgen-dependent male sexual differentiation in humans vs. mice yet, both models support the essential role of LH in the later stages of follicle development and ovulation.
Although the first clinical description of a patient with isolated FSH
deficiency dates back to 1972 (17), the molecular basis of
this condition was not confirmed until almost 20 yr later
(18). A patient with primary amenorrhea, sexual
infantilism, eunuchoidism, low FSH/estradiol levels, and high LH levels
was found to harbor a homozygous deletion at codon 61 of the ß
subunit of FSH (18), predicted to result in a truncated
protein that is unable to associate with the -subunit. Treatment
with recombinant FSH resulted in ovulation and conception,
demonstrating the critical role of FSH in normal follicular
development, ovulation, and fertility. A second patient demonstrated
dramatic increases in androgen levels after exogenous FSH and hCG,
demonstrating the critical role of FSH in LH-induced androgen
production (19).
In contrast to women, one might predict that males bearing FSHß mutations would not demonstrate signs of androgen deficiency. However, naturally occurring human mutations have shown otherwise. The first proband with an FSHß mutation (Cys82Arg) presented with normal puberty and azoospermia, whereas the second (Val61X) presented with an absence of pubertal development and azoospermia (20, 21). The Leydig cell hypofunction in this second patient suggests that FSH, or some FSH-dependent Sertoli cell factor, is necessary for androgen production in men. In contrast to the azoospermia observed in the human male FSHß knockout, mice with similar mutations are fertile despite a decrease in testicular size and reduced sperm counts (22). Therefore, although FSH appears necessary for normal Sertoli cell development and the initiation (as opposed to the maintenance) of spermatogenesis in men, it may not be essential in mice.
While the pathophysiology of FSHß mutations were elucidated through the study of individual patients, a large scale linkage dysequilibrium approach was used to identify the genetic determinants of hypergonadotropic hypogonadism in Finnish women with primary amenorrhea, elevated gonadotropins, and a normal karyotype (23). A mutation was identified in the extracellular domain of the FSH receptor that led to impaired ligand binding and reduced cAMP stimulation. Ovarian biopsies revealed a full range of primordial follicles demonstrating that FSH is not necessary for normal follicular development before the antral stage. Female FSH knockout mice are also infertile with follicular development similarly arrested at the preantral stage (24, 25).
Although there is general concordance between female patients with FSHß and FSH receptor mutations in terms of their demonstration of primary amenorrhea and arrested follicular development, a larger spectrum of discrepancies exist in men. Five men with FSH receptor mutations have been reported, all of whom were normally masculinized but had diminished testicular volumes (26). In contrast to the men with FSHß mutations, both of whom were azoospermic, men with FSH receptor mutations demonstrate variable spermatogenic activity, ranging from oligospermia to normal sperm counts. Two of the five men had previously fathered children. Male mouse knockouts of the FSH receptor confirm the critical role of the LH/Leydig cell axis in spermiogenesis—fertility is present, albeit in the setting of abnormal spermatogenesis (24, 25). Interestingly, however, despite high FSH concentrations, testicular inhibin A and B concentrations were not significantly different from those of normal littermates, suggesting that the regulation of this protein may also be less dependent on direct stimulation from FSH than was previously hypothesized (25). Taken together, studies of FSH receptor mutations have raised several new questions about the role of FSH in ovarian development, male fertility, and "feedforward" regulation of intratesticular paracrine factors.
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Adrenal hypoplasia congenita and DAX1 |
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TopIntroductionInactivating mutations of the...Adrenal hypoplasia congenita and...Kallmann syndrome and the...References |
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Boys with AHC typically present with adrenal insufficiency in early infancy or childhood and hypogonadotropic hypogonadism at the time of puberty. However, normal serum gonadotropin and testosterone levels have been demonstrated in newborn males with AHC and a confirmed DAX1 mutation (30, 31). These observations suggest that the hypogonadotropism of AHC, rather than being congenital in nature, develops over time in some target organs. Although greater awareness of this disorder has led to more timely treatment of the hypogonadism in these patients, long-term data on their gonadal function and fertility are lacking. However, studies in both the human and the mouse suggest that, unlike other forms of hypogonadotropic hypogonadism in which exogenous gonadotropins or pulsatile GnRH can stimulate sperm production, patients with AHC can be resistant to such therapies. Testicular biopsy from a 27-yr-old male with AHC and a confirmed DAX1 mutation (501delA) revealed rare spermatogonia and Leydig cell hyperplasia (32). This patient remained azoospermic despite 3 yr of progressive doses of hCG and Pergonal and steady increments in testicular size. Targeted disruption of the mouse homolog of DAX1, Ahch, reveals relatively normal testes at birth but progressive epithelial dysgenesis and sloughing of germ cells by 10 weeks, and complete loss of germ cells by 14 weeks (33). Leydig cell hyperplasia and hypertrophy are also observed. Although the hypogonadism of human AHC has traditionally been attributed to a deficiency in gonadotropin secretion, the mouse knockout model, coupled with these human observations, reveals that DAX1 may play a critical role in Sertoli cell function and that restoration of a normal trophic hormonal milieu cannot overcome this defect. Also of note, despite the sex-reversal that occurs in Ahch overexpression, female mice Ahch knockouts exhibit normal sexual differentiation, ovulation, and fertility, establishing this nuclear receptor as a critical factor for testicular function postnatally.
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Kallmann syndrome and the KAL gene |
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TopIntroductionInactivating mutations of the...Adrenal hypoplasia congenita and...Kallmann syndrome and the...References |
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Although Kallmann syndrome is genetically heterogenous (35) and the autosomal genes responsible for this condition remain unknown, the X-linked gene (KAL) has been cloned and encodes a secreted protein, anosmin (36, 37). The amino-terminus contains a four-disulfide core domain characteristic of proteins with antiserine protease activity and neurophysins (38). The carboxy-terminus contains three fibronectin III (FNIII) repeats characteristic of extracellular matrix molecules, a protein family well known for its role in neuronal migration (39). Mutation analysis of patients with X-linked Kallmann syndrome has revealed a spectrum of coding sequence abnormalities (40, 41). However, almost all single point mutations occur in the FNIII domains, suggesting that these domains are critical for the functions of the KAL protein and hence to GnRH neuronal migration (41).
As KAL’s murine homolog has yet to be identified, expression studies of this gene had to be performed in the chick. In this animal model, KAL transcripts are not detected until the later stages of embryonic development and adulthood (42, 43). KAL expression is triggered when the olfactory bulb begins to differentiate, increases as the bulb takes on its characteristic architecture, and continues into adulthood (42, 43). Although anosmin appears to induce neurite outgrowth in a cell-specific manner (44), the mechanism by which it mediates its neuronal targeting—adhesive substrate, chemoattractant, or guidance factor—is not yet understood. What appears clear is that when there are defects in the KAL gene and, by extension in its protein product, anosmin, GnRH neurons are unable to migrate along their olfactory "trellis." Therefore, the hypogonadotropic hypogonadism of Kallmann syndrome appears to be a by-product of a primary abnormality in olfactory development.
Although hypogonadotropic hypogonadism and anosmia are the signature features of Kallmann syndrome, several other somatic anomalies are associated with this condition, including unilateral renal agenesis, synkinesia or mirror movements, hearing loss, midline facial defects, and bony abnormalities (40, 45). In the chick, KAL expression has been identified in the Purkinje fibers of the cerebellum, oculomotor nucleus, mesodermal derivatives, and mesenchyme (42, 43). In the human, the KAL-encoded protein, anosmin, is a temporally and regionally defined extracellular component of interstitial matrices and basement membranes during embryonic development (46). It is found in the extracellular matrices of various structures including bronchial tubes, mesonephric tubules, ureteric bud, digestive tract, large blood vessels, and inner ear. Anosmin was not identified in the olfactory epithelium nor in the fronto-nasal mesenchyme but was found in the anlagen of the olfactory bulbs and the primitive cerebral hemispheres, confirming its role in the later events of olfactory nerve migration/olfactory bulb differentiation. Thus, the theme of genes/proteins key to reproduction being used in other nonreproductive functions is highlighted by Kallmann syndrome.
From these experiments of nature in the human and targeted disruption at the bench, several themes emerge. Mutations in gonadotropin subunits and their receptors reveal that the relationships between gonadotropin stimulation and sex steroid production, testicular volume, follicle development, and fertility are much more complex than previously thought. Genes such as DAX1 that have traditionally been implicated in secondary hypogonadism actually have direct gonadal effects in the regulation of spermatogenesis; thus, their hypogonadotropism may mask their primary gonadal effects. Certainly, there will be additional genes that act at all levels of the hypothalamic-pituitary-gonadal axis, and the description of new clinical disorders should continue to elucidate their presence. Finally, gene defects that cause abnormalities in GnRH neuronal migration play important roles in axonal targeting beyond the reproductive system, broadening our thinking about reproductive phenotypes as parts of larger, developmental syndromes. Sophisticated knockout models and gene rescue experiments will provide additional and exciting new avenues of investigation in the coming years.
Received March 16, 2001.
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References |
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TopIntroductionInactivating mutations of the...Adrenal hypoplasia congenita and...Kallmann syndrome and the...References |
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