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Andrology Laboratory, ANZAC Research Institute, Concord Hospital, University of Sydney, New South Wales 2139, Australia
ABSTRACT
Although androgens and the androgen receptor (AR) have defining roles in male reproductive development and function, previously no role in female reproductive physiology beyond testosterone (T) as the precursor in estradiol (E2) biosynthesis was firmly established. Understanding the role and specific mechanisms of androgen action via the AR in the ovary has been limited by confusion on how to interpret results from pharmacological studies, because many androgens can be metabolized in vivo and in vitro to steroids that can also exert actions via the estrogen receptor (ESR). Recent genetic studies using mouse models with specific disruption of the Ar gene have highlighted the role that AR-mediated actions play in maintaining female fertility through key roles in the regulation of follicle health, development, and ovulation. Furthermore, these genetic studies have revealed that AR-mediated effects influence age-related female fertility, possibly via mechanisms acting predominantly at the hypothalamic-pituitary axis in a dose-dependent manner. This review focuses on combining the findings from pharmacological studies and novel genetic mouse models to unravel the roles of ovarian androgen actions in relation to female fertility and ovarian aging, as well as creating new insights into the role of androgens in androgen-associated reproductive disorders such as polycystic ovarian syndrome.
androgen receptor, fertility, follicular development, ovary
Androgens mediate their action primarily via the androgen receptor (AR), a member of the nuclear receptor superfamily encoded by an X chromosomal gene [1]. Classically, androgen-activated AR exerts its biological effects by stimulating target genes via a sequence of processes, including ligand binding, homodimerization, nuclear translocation, DNA binding, and complex formation with co-regulators and general transcription factors [2].
Although the pivotal role of androgens in male reproductive function [2] and the obligatory role of testosterone (T) as an estradiol (E2) precursor in females are well understood [3, 4], the direct involvement of androgens in female reproductive physiology remains controversial. Clinical evidence for a physiological role of androgens in human female health comes from findings that women with complete adrenal failure exhibit clinical benefits after replacement of dehydroepiandrosterone (DHEA) in some [5, 6], but not all [7, 8], studies, and women respond well to the pharmacological use of T and DHEA to treat female androgen insufficiency [9, 10]. In addition, androgens are associated with female reproductive pathology, notably polycystic ovarian syndrome (PCOS), a common condition that features excessive ovarian androgen production and causes infertility, acne, and hirsutism [11].
Further evidence for the role of androgens in female reproductive physiology arises from in vitro pharmacological observations that several androgens, including T, androstenedione (A4), and dihydrotestosterone (DHT), can stimulate follicle growth and development [12, 13]. However, many androgens, such as T and A4, can be aromatized into corresponding estrogens, such as E2 and estrone (E1), which causes confusion when dissecting the precise molecular mechanism(s) causing the effect. Traditionally, DHT, a nonaromatizable androgen, has been used to experimentally distinguish between the estrogenic and androgenic actions mediated by the estrogen receptor (ESR) and the AR, respectively. However, this may be problematic, as DHT is enzymatically reduced into the 5alpha-androstanediols, 3alpha-diol and 3beta-diol, which are both unable to bind AR, and hence biologically inactive as an androgen. Yet, these DHT-derived diols can either activate the ESR (3beta-diol) to exert indirect steroidal effects or indirectly feed the AR pathway (3alpha-diol). The latter, 3alpha-diol, has almost no affinity for steroid receptors [14, 15], but can act as an androgen precursor reservoir because it is readily back-converted to DHT [16–18]. On the other hand, 3beta-diol is formed by a virtually irreversible process [19, 20] and has a specific high affinity for estrogen receptor beta (ESR2), but only negligible binding affinity (RBA value <0.1) for estrogen receptor alpha (ESR1) [21, 22]. Hence, pharmacological studies relying on nonaromatizable DHT cannot fully discount the possibility that this potent androgen may be converted into other steroids with potential ESR-mediated actions, confounding potentially androgenic responses (Fig. 1).
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Studying females homozygous for an inactivated AR (Ar–/–) has provided a key insight into the role of AR-mediated effects on the ovary. However, Ar–/– females cannot be produced by natural mating because hemizygous males with an inactive AR (the classical complete androgen insensitivity syndrome [CAIS], formerly known as testicular feminization syndrome [Tfm]) are sterile. Therefore, the first research models for female androgen insensitivity utilized the naturally occurring but rare XO female mice in which the X chromosome bore the ArTfm mutation [23], and the homozygous ArTfm/ArTfm female mice created by breeding males embryonically chimeric for hemizygous ArTfm/Y with females heterozygous for ArTfm [24]. Both original models required complex production methods that were inefficient at providing propagatable lines of mice for analysis. More recently, homozygous Ar–/– female mice have been produced efficiently using the Cre/LoxP system [25–28]. Using these genetic models, there is now definitive proof of a role for AR-mediated actions in follicle development (summarized in Fig. 2).
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AR mRNA and Protein Expression
Ar mRNA and AR protein are present in the oocyte, granulosa cells, and theca cells of rodent ovaries [29–33]. Ar mRNA demonstrates intense staining in rat theca cells, whereas granulosa cells have moderate labeling [29]. In a similar study, Ar mRNA is predominantly located in rat granulosa cells, with expression most abundant in small follicles and decreasing as the follicle develops [30]. Correspondingly, AR immunostaining, although not as quantitative, progressively declines in the mural granulosa cells of later-stage antral follicles, but a few mural granulosa cells bordering the antrum and the cumulus cells maintained strong AR-positive staining [31]. In the bovine and ovine ovary, AR mRNA is present in granulosa and theca cells, with the most intense expression in granulosa cells of preantral and antral follicles, but is absent in bovine primordial follicles [34, 35]. In porcine follicles, AR mRNA expression is highest in the granulosa cells until the antral stage and then decreases with follicle maturation [36, 37]. AR protein is present in the oocyte, granulosa cells, and theca cells of preantral and antral follicles within ovine and porcine ovaries [34, 38]. Within the primate ovary, AR mRNA and AR protein expression is most abundant in granulosa cells of preantral and antral follicles [39, 40]. AR mRNA is expressed in preantral and antral follicle theca cells, but to a lesser degree than granulosa cells [39]. Furthermore, AR mRNA present in luteinized granulosa cells [41] increased after ovulatory stimulus (hCG), whereas ESR2 mRNA decreased [42], indicating a switch in steroid receptor-mediated actions during preovulatory events. However, little or no specific AR immunostaining has been detected in theca cells or oocytes, and AR immunostaining was either low or absent in preovulatory follicles [40]. Finally, in humans, AR protein has been localized to the theca cells of preantral follicles [43], granulosa cells of antral follicles [44], and most intensely in granulosa and theca cells of dominant follicles [45].
Although AR expression is present throughout most stages of follicular development (Fig. 3), distinct patterns of AR expression have been shown within follicles of different mammalian species, which may highlight the importance of the follicular developmental stage and species-specific differences for AR activity.
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Rats treated with FSH exhibit a decreased abundance of ovarian Ar mRNA, which is further down-regulated by LH cotreatment, consistent with declining Ar expression in maturing granulosa cells of gonadotropin-stimulated preovulatory follicles [32]. This Ar decrease is coupled with increased Cyp19a1 (P450arom) mRNA expression, suggesting a switch from predominantly AR-mediated to ESR-mediated effects [32]. Ar expression in rat preantral granulosa cells can be down-regulated by the addition of DHT, but is restored by FSH treatment [30]. Conversely, the down-regulation of Ar expression in rat preovulatory follicles is not directly caused by reduced FSH activity [30], which implies that the regulation of Ar expression is developmental status dependent. Oocyte-secreted factors have been proposed to influence AR expression, because during the later stages of follicular development a gradient of AR immunostaining exists with a decrease in AR expression proceeding towards the antrum, while the cumulus cells that are closest to the oocyte maintain intense AR staining [31]. This also implies that within the ovary AR expression may play an important role in local control mechanisms during follicle development.
In primates, AR mRNA expression in preovulatory follicle granulosa cells increases 24 and 36 h post-hCG treatment, suggesting a role in periovulatory events [42]. Furthermore, in small antral follicles, in vivo treatment with T increases AR mRNA in granulosa and theca cells, and its expression is positively correlated with FSH receptor (FSHR) mRNA and proliferation, but negatively correlated with apoptosis [39]. However, it is unclear whether these effects are AR or ESR mediated as T may have been aromatized to E2. In spite of this, the steep decline of AR observed in maturing follicles of several species may be critical in the establishment of follicle dominance [40]. Furthermore, since T can enhance FSH-induced actions, such as aromatase activity, cAMP formation, and action [46, 47], the down-regulation of AR in mature follicles of some species may be critical in reducing androgenic effects during follicle selection and follicular atresia.
PHARMACOLOGICAL STUDIES OF ANDROGEN ACTION ON FOLLICLE DEVELOPMENT
Exogenous androgens exert both inhibitory and stimulatory effects at different developmental stages throughout the process of follicular development (Fig. 3). However, the majority of pharmacological studies are limited in their ability to differentiate whether the effects observed are solely AR mediated or are also due to androgen conversion to other steroids with the potential to exert indirect actions such as via the ESR. Hence, some of the conflicting data on androgens causing both inhibitory and stimulatory effects may be attributed to local androgen metabolism.
Androgen Control of Key Regulators of Follicular Development
In vivo treatment of primate ovaries with T increased FSHR mRNA expression in primary follicles [48]; similarly, DHT increased FSHR expression in preovulatory follicles in gilts [49]. However, these studies do not account for the possibility of androgen conversion and hence indirect androgen actions. More conclusively, though estrogens (E1 and E2) exerted no stimulatory effects, T increased murine follicle responsiveness to FSH [12], and A4 stimulated FSH-mediated differentiation of bovine granulosa cells, indicated by an increase in aromatase activity and E2 production [50], presumably due to the increased postreceptor cAMP response [47].
The intraovarian insulin-like growth factor (IGF) autocrine/paracrine system plays an essential role in regulating ovarian follicular development. In vivo treatment with T or DHT promotes an increase in IGF1 and IGF1 receptor (IGF1R) mRNA in primate ovaries [51, 52]. Likewise, in vitro treatment of porcine mural granulosa cells with DHT enhances IGF1-stimulated proliferation and the mitogenic effects of growth differentiation factor 9 (GDF9) in the presence of IGF1 [53, 54], which are blocked by the presence of an AR antagonist (hydroxyflutamide). Hence, androgens acting via the AR may regulate the expression and action of one or more key ovarian growth factors during different stages of follicle growth.
Deciphering the Effects of Androgens on Follicle Development
Actions of androgens alone. Administration of T or DHT to female rhesus monkeys and pregnant ewes (prenatally treated fetuses) stimulates primordial follicle initiation and increases growing follicle numbers and overall follicle survival [52, 55–57]. Furthermore, prenatal T-treated ewes exhibit an increase in large antral follicles (diameter 7 mm) and follicular persistence, whereas prenatal DHT-treated ewes display an increase in the number of small antral follicles (diameter 3–4 mm). These findings suggest that androgenic actions play a role in follicle initiation and early growth, whereas estrogenic actions play a more dominant role in late follicle development and follicular persistence [58]. Conversely, in vivo treatment of rats with DHT inhibits ovarian follicular development and function [59]; similarly, addition of A4 inhibits mouse preantral follicle growth and E2 production in culture [60]. Administration of T or DHT during the follicular phase increases preovulatory and corpora lutea numbers observed within porcine ovaries, supporting a positive effect of androgens on ovulation [49, 61]. However, in these studies it is unclear whether T and A4 are acting directly on AR or indirectly as a substrate for aromatization to an estrogen or conversion of DHT to 3beta-diol, which could potentially mediate effects via ESR2. Hence, these studies fail to exclude indirect actions of androgen through local conversion to other steroids. Another limitation of these in vivo approaches [52, 56] is that the administration of T or DHT was not carried out at a specific stage of the estrous cycle, and gonadotropin levels were not measured or controlled. Although the majority of effects on follicle growth are during early stages of development and generally thought to be gonadotropin independent, this treatment is likely to exhibit feedback effects on pituitary gonadotropin secretion. Therefore, it is difficult to conclude whether the observed ovarian effects of exogenous androgen treatment are direct or due to changes in gonadotropin levels. Administration of T or DHT in the porcine model was carried out at a more specific stage of the estrous cycle [49, 61]; however, gonadotropin levels again were not measured. Hence, it is not clear if all the follicular effects observed can be attributed conclusively to direct androgen actions.
Alterations in intraovarian/intrafollicular levels of steroids may be of importance in determining the response and fate of individual follicles. Intraovarian steroidogenic properties in rodents [62], cows [63], and humans [64] are related to follicular developmental stage and circulating gonadotropin levels, indicating that local androgen:estrogen ratios play an important role in determining follicular fate. During follicular growth, changes in follicular fluid steroid levels have been correlated with follicle health and stage of development [65–68]. In humans, there is a predominant pattern of steroids in the follicular fluid that relates to follicular development, in which small antral follicles are androgenic, large antral follicles are estrogenic, and preovulatory follicles are progestagenic, whereas atretic follicles of all sizes exhibited an androgenic pattern of steroids in their follicular fluid [65]. Furthermore, the human oocyte-cumulus complex (OCC) can produce androgens, estrogens, and progesterone (P4), so is capable of modifying its own steroidal microenvironment [69]. Thus, changes in intrafollicular steroid concentrations may influence the oocyte directly and indirectly via the granulosa and cumulus cells.
Androgen and estrogen actions alone and in the presence of AR antagonists and aromatase blockers. Some pharmacological studies have provided strong support for AR-mediated actions in follicle development by describing the effects of androgens and estrogens both alone and in the presence of AR antagonists or aromatase blockers. Failure of cultured preantral mouse follicles to develop to preovulatory follicles in the presence of anti-androgen antibodies and AR antagonist (bicalutamide) treatment [13] implies that AR-mediated actions are important in the early stages of follicular development. Bovine primary to secondary follicle transition is stimulated by T [70] but not E2, and the addition of an AR blocker (flutamide) inhibits the observed stimulatory effect, indicating that the effect is due to a direct AR-mediated action, not the aromatization of T to E2. Similarly, mouse preantral follicular development is enhanced after in vitro culture with T, DHT, A4, DHEA, or DHEA sulfate, with follicles undergoing rapid granulosa cell proliferation and amplified responsiveness to FSH [12]. Again, stimulation of follicle development observed could not be due to aromatization because addition of an AR antagonist (flutamide) blocked the growth effect, whereas estrogens (E1 or E2) alone had no effect on growth. Furthermore, androgens in the presence of aromatase inhibitors (fadrozole) did not block the enhanced follicular growth. Nevertheless, this pharmacological approach still fails to account for the possible conversion of DHT to 3beta-diol, and the findings depend on the specificity of the pharmacological agents used.
During the later stages of follicle growth, T stimulates in vitro oocyte maturation by completely reversing the IBMX inhibition of germinal vesicle breakdown, indicating that T is capable of overcoming the elevated intracellular cAMP [33]. Moreover, addition of AR antagonists (flutamide and hydroxyflutamide) suppresses the T-mediated response, whereas the transcriptional inhibitor actinomycin D does not, suggesting that some AR-mediated actions may not require transcription [33]. Androgens have been reported to induce nongenomic effects, but characterization of their role in vivo requires much clarification [71, 72]. Interestingly, E2 exerts a beneficial effect on oocyte cytoplasmic maturation via nongenomic effects, which can be antagonized by the addition of A4; however, A4 alone has no effect [73]. DHT, T, and E2 potentiate the FSH-induced inhibition of meiotic resumption in mouse OCCs [74], and in contrast to the previous study [33], T alone inhibited oocyte meiotic maturation and embryonic development in a dose-dependent manner [75], indicating that T may act to maintain high cAMP levels. Androgens also improve the ovulatory response of superovulated mouse ovaries, whereas antiandrogens (cyproterone and cyproterone acetate) at high doses decreased the number of ovulations [76]. Antiserum to T reduces ovulation rates of rat ovaries, whereas treatment with T or DHT restores ovulation in the presence of antiserum to P4 (which blocks ovulation) [77]. However, steroid reduction in primates during the gonadotropin surge, which abolishes ovulation, can not be restored by the addition of DHT [78]. The opposing findings for the role of androgens in oocyte maturation and ovulation outlined here highlight the need for further detailed studies in this area.
In summary, exogenous androgens appear to enhance follicle development. However, because the findings depend on the specificity and effectiveness of the blockers used and steroid metabolism, whether these effects are mediated solely via the AR requires further critical verification. Nevertheless, the changing spatial and temporal pattern of AR expression, stage of follicle development, and possibly androgen concentration during follicle growth may all play important roles in regulating the extra- and intraovarian mechanisms ultimately determining follicular fate.
REGULATION OF FOLLICULAR ATRESIA BY ANDROGENS
Follicular atresia is thought to occur through an apoptotic mechanism [79, 80], the best characterized form of physiological or programmed cell death [81]. Apoptosis in ovarian follicles is regulated by different factors at different developmental stages.
Indirect Modulation of Follicle Atresia by Androgens
Androgens are required as a substrate for the FSH-dependent process of estrogen biosynthesis. Both FSH [82] and E2 [83] are known to act as follicle survival factors, with continued follicular growth from the late preantral/early antral stage dependent upon FSH stimulation and E2 production. Hence, as steroid substrates, androgens indirectly protect the developing follicle from undergoing atresia. Treatment of primate granulosa cells from small antral follicles with FSH and T or DHT leads to a dramatic increase in steroidogenesis, whereas androgen treatment of granulosa cells from large antral follicles leads to an inhibition of FSH-stimulated aromatase activity [84]. Hence, androgens augment the FSH responsiveness of somatic cells in a developmentally stage-dependent manner, which may contribute to the mechanisms controlling whether a follicle continues to grow or undergo atresia.
Direct Actions of Androgens on Follicle Atresia
In vivo, T induced follicular atresia defined by both oocyte degeneration and granulosa cell pyknosis [85], and DHT and T increased somatic cell atresia in rat ovaries [86]. T antagonized the antiapoptotic effects of E2 in rat granulosa cells in early antral and preantral follicles [83], and an abnormally high level of AR protein expression in Esr2 knockout mice (Esr2tm1Unc/ Esr2tm1Unc) was associated with increased somatic cell atresia in late antral follicles [87]. Exposure of immature murine oocytes to T reduced their ability to mature and undergo normal embryonic development [75], suggesting that these oocytes may be programmed to undergo cell death; however, just prior to ovulation, AR-mediated actions are thought to be important due to the maintenance of AR expression in cumulus cells of the OCC [31].
In summary, androgens may regulate follicular atresia both directly and indirectly. However, these results highlight that of most importance in maintaining follicle health is the appropriate timing of androgen exposure to the developing follicle. Furthermore, as with follicle development, deducing the exact mechanisms can be problematic due to androgen conversion, and pharmacological approaches are limited by the specificity and effectiveness of drugs used.
FEMALE AR-DEFICIENT MOUSE MODELS
The production of the homozygous female Ar–/– mouse model has provided a fundamental advance in unraveling the roles of AR-mediated androgen action in female reproductive physiology. The loss of functional AR has highlighted the key role AR-mediated actions play in maintaining female fertility.
The original models for female androgen insensitivity, the naturally occurring but rare XtfmO [23] and the homozygous ArTfm female mouse [24], exhibited increased follicle atresia and reduced follicle numbers, but AR-mediated androgen action was qualitatively not essential for ovulation, mating, pregnancy or lactation [23, 24, 88]. However, little further work was carried out using these models presumably because of the difficulties in producing sustainable numbers of mice. More recently, a regular supply of female Ar–/– mice has been generated efficiently using the Cre/LoxP system [25–28, 89]. Models were created by a targeted deletion of exon 1 (Artm1Ska/Artm1Ska, denoted hereafter as ArEX1–/–) [27], exon 2 (Artm1.1Chc/Artm1.1Chc, denoted hereafter as ArEX2–/–) [25, 26], or exon 3 (Artm1.1Jdz/Artm1.1Jdz, denoted hereafter as ArEX3–/–) [28] of the Ar. The ArEX1–/– and ArEX2–/– models relied upon major loss of the AR protein due to insertion of premature stop codons resulting in the deletion of 7 or 6 of the 8 exons, respectively. By contrast, the ArEX3–/– approach generated an in-frame excision of exon 3 alone, which encodes the second zinc finger essential for DNA-binding. Therefore, ArEX3–/– mice retain a mutant AR protein that is nonfunctional as a direct nuclear transcription factor. Modified AR proteins produced by point mutations or a deletion in exon 3 have normal androgen-binding affinity and nuclear localization, but a markedly reduced DNA-binding affinity and an inability to transactivate androgen-responsive reporters in vitro [90–92]. Males hemizygous for ArEX3–/– confirmed the abolition of classic genomic AR function by exhibiting the CAIS (Tfm) phenotype [93], as have the major deletions produced by the other models [25, 94]. Comparison between the different models allows a detailed analysis of AR-mediated actions on female fertility and ovarian follicular growth and health (Fig. 2).
All Ar–/– female mouse models described to date are subfertile with fewer pups per litter and decreased follicle health [25–28], but exhibit normal follicle populations at least up to 16 wk of age [26–28]. Older females with a complete loss of AR protein (ArEX1–/–) exhibit accelerated follicle depletion [27], but this is not apparent in the ArEX3–/– model, which maintains a minimally truncated but inactive AR protein. Furthermore, the heterozygous ArEX3+/– mice exhibit an age-dependent reduction in pups per litter, indicating a significant Ar gene dosage effect on female fertility [28].
Ovarian Fsh and Igf1r expression is noticeably reduced in ArEX2–/– mice [26], indicating that follicle growth may be impaired. In contrast, follicle growth rates are not altered in the ArEX3–/– model, indicating that classical AR-mediated actions do not appear to play essential roles in follicle growth [28]. Oocyte health is compromised after total loss of AR protein (ArEX2–/–), with oocytes in preovulatory follicles exhibiting a loss of cumulus cell contact during ovulation and reduced expression of genes required for cumulus expansion, such as hyaluronan synthase 2 (Has2) and tumor necrosis factor-
-stimulated gene 6 (Tsg6) [26]. Moreover, microarray analysis of ArEX1–/– ovaries indicated a reduced expression of several genes involved in the oocyte-granulosa cell regulatory loop [27], including KIT ligand (Kitl), bone morphogenetic protein 15 (Bmp15) and Gdf9. However, it remains to be clarified whether this decrease in expression is due directly to the loss of AR or a secondary consequence of the differences in follicle populations present. ArEX3–/– oocyte viability appears unaffected, with no disassociation of cumulus cells in preovulatory follicles (Walters, unpublished data), normal fertilization rates, and early embryonic development [28], suggesting that the adverse effects on follicle and oocyte health and development observed in other AR-deficient models may be partly a secondary consequence of the complete absence of the AR protein. In other words, the ArEX3–/– females may retain an as yet undefined AR activity that is independent of direct AR DNA-binding mediated transactivation.
Defective follicle development during the final stages of follicular growth and ovulation is displayed in all Ar–/– female models, with reduced ovulation of oocytes and fewer corpora lutea formed [26–28]. Interestingly, in the ArEX3–/– model, the reduced ovulation rates are overcome by gonadotropin hyperstimulation [28]. This result, together with delayed time to first litter [28] and abnormally long estrous cycles [26], suggests a predominantly extraovarian defect in the hypothalamic-pituitary regulation. Further studies are required to define whether the primary androgenic effects on follicle development and fertility are intra- and/or extraovarian. However, the use of these distinct Ar–/– models has confirmed that AR-mediated actions do play an important role in maintaining female fertility. We believe that differences in results from the three models do not reflect biological differences in the role of the AR, but rather differences in the generations of the three models and consequent secondary effects. All models produced an equally subfertile female; however, the female mice with a deletion in exon 1 and 2 of the Ar exhibit more severe phenotypes than the model with an in-frame deletion of exon 3, which still allows the production of a transcriptional inactive protein. We hypothesize that these more dramatic effects on ovarian function are a secondary consequence of the loss of the protein, which may interact with co-regulators and other transcription factors beyond that of the AR, rather than a selective direct loss of AR transcriptional activity.
HUMAN IMPLICATIONS OF DIRECT ANDROGEN ACTION IN THE OVARY
Determinants of Female Ovarian Aging and Follicle Depletion
As menopause occurs when depletion of follicle stock reaches a critical threshold, the rate of atresia, at least in part, dictates the functional lifespan of the ovary and hence the age of menopause [95]. Previous animal studies show that androgens may enhance [83] or inhibit [56] follicular atresia and can influence follicle atresia by manipulating somatic cell apoptosis and/or oocyte degeneration. ArEX1–/– females with loss of AR protein exhibit increased atresia, which is positively correlated with an early decline in follicle numbers leading to accelerated ovarian failure [27]. However, the claimed relationship between inactivating Ar mutations and premature ovarian failure remains to be verified, as to date there is no evidence of Ar mutations in women with premature ovarian failure, a condition associated with chromosome abnormalities, particularly involving the X chromosome [96]. Because the observed depletion of follicles in the ArEX1–/– model is not apparent in the ArEX3–/– model, which retains a mutant but inactive AR protein, it is doubtful that follicle depletion depends solely on AR activation. Indeed, we suggest that the premature loss of follicle reserve observed in the ArEX1–/– model may be a secondary consequence of the loss of protein, which may lead to the disruption of other pathways beyond that of AR transcription.
Furthermore, the finding that heterozygous ArEX3+/– mice had no accelerated loss of follicle populations but a significant age-related reduction in fertility [28], suggests that quantitative variations in AR activity via gene dosage play a role in determining female fecundity and ovarian aging. In addition to haploinsufficiency, a disruption in AR-mediated actions that acts to interrupt hypothalamic-pituitary signaling is indicated by the observed reduction in ovulation rates in the AREX3–/– mice, which can be rescued by gonadotropin hyperstimulation. Hence, a central extraovarian mechanism regulated by AR may play a role in determining the timing of onset of AR-related reproductive disorders and/or reproductive aging. Obviously, fundamental differences between human and rodent reproductive aging, such as a lack of a true menopause observed in rodents, make comparison between the two species difficult. However, despite these limitations, there are similarities in how reproductive aging is regulated by the hypothalamic-pituitary axis, so parallels can be drawn using the mouse as a practical experimental model. Ar is expressed in the mouse brain and regulated by T and E2 [97]; therefore, AR signaling has the potential to influence feedback mechanisms regulating the hypothalamic GnRH and pituitary LH and FSH release, including the ovulatory LH surge. Therefore, we propose that AR-mediated androgen actions acting predominantly at the hypothalamic-pituitary axis may contribute to the timing of the onset of AR-associated reproductive disorders and/or ovarian aging within the population.
Although the complete homozygous inactivation of AR cannot occur naturally in women, the finding that heterozygous ArEX3+/– mice exhibit an age-dependent reduction in fertility [28] may have implications for the reproductive performance of women who are carriers of CAIS AR mutations. The ArEX3+/– model predicts that mothers of males with CAIS who are obligate heterozygotes for the same mutation may have curtailed reproductive function, particularly at an older age. Hence, studies examining whether such women exhibit a reduction of age-related fertility and/or relationship to the timing of spontaneous follicular exhaustion (age at menopause) would be of interest. It may also be speculated that these obligate CAIS carriers may be less sensitive to androgens than AR+/+ women, and thereby less susceptible to the prevalence of androgen-associated diseases such as PCOS.
PCOS is characterized by arrested follicular maturation with excessive ovarian production of androgens, but the origins and underlying mechanism of PCOS remains unclear [11]. The findings that human theca interna cells from the PCOS ovary produce 20 times more A4 than those from normal ovaries [98], which persists over a long-term culture [99], and treatment with the AR antagonist flutamide improved fertility in some women with PCOS [100, 101], are consistent with a role for AR-mediated actions. PCOS has been proposed as having its origin in fetal life as animal models (monkeys, sheep, rodents) with prenatal exposure to high doses of androgens leading to the development of many of the characteristic features of PCOS [102]. Young ArEX3–/– mice exhibit transient elevated intraovarian T levels, which is associated with poor follicle health and disrupted ovulation [28], raising the hypothesis that even transient abnormally high levels of ovarian androgens may initiate lasting effects on the developing follicle.
Variations in CAG repeats within exon 1 of the AR have been linked to the prevalence of PCOS [103, 104]. Paradoxically, both short [104] and long [103] CAG repeat lengths in different sub-populations have been associated with the prevalence of PCOS; therefore, it is unclear whether variations in the CAG repeats lead to higher prevalence of PCOS or act by modulating pre-existing androgen-related diseases [105].
Genetic studies investigating the role of androgens in female reproductive physiology have complemented and extended the observations from previous pharmacological approaches by confirming the role for AR-mediated actions and providing new insights into the role of androgens in female reproduction. These studies have revealed that androgen action does play a role in regulating follicle development and ovulation, and AR-mediated actions may underlie new facets of the hormonal regulation of female fertility. In particular, the use of Ar–/– female mouse models has revealed that AR function is essential to maintaining age-related female fertility, notably through optimizing the conditions for follicular growth, particularly during the final stages of development and especially ovulation. Furthermore, the ArEX3–/– female mouse model identified that reduced ovulation rates could be overcome by gonadotropin hyperstimulation, suggesting a disruption at the hypothalamic-pituitary axis [28]. In addition, the heterozygous ArEX3+/– mouse [28] exhibited an age-dependent reduction in fertility. Taken together, these results indicate that AR-mediated actions may play an important role in maintaining female fertility predominantly via the hypothalamic-pituitary axis in a dose-dependent manner. A better understanding of the role of androgens in female reproductive physiology may allow us to elucidate further AR-mediated mechanisms governing the rate of follicular development, and hence lead to novel approaches in the treatment of age-related infertility as well as androgen-associated disorders, such as PCOS.
Correspondence: 1David J. Handelsman, ANZAC Research Institute, Sydney, NSW 2139, Australia. FAX: 61 2 9767 9101; e-mail: djh{at}anzac.edu.au
Received: 5 July 2007.
First decision: 15 August 2007.
Accepted: 2 November 2007.
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