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ESTROGENS AND MALE REPRODUCTION
Vincenzo Rochira, Fabbi Matteo, Valassi Elena and Cesare Carani
January 7, 2003 Inde
INTRODUCTION
The intriguing concept that a role for estrogens exists in male reproduction has recently been recognized in the field of endocrinology (1, 2). It has developed from observations and studies that have been performed over the last 10 years. In particular, the development of lines of male transgenic mice lacking functional estrogen receptors or a functional aromatase enzyme have shed new light on the role for estrogens in male reproduction (3). Concomitantly, the discovery of mutations in both the human estrogen receptor alpha (4) and aromatase (5) genes have reinforced the idea that estrogens play a key role in the human male reproductive system.
Previously a role for estrogen action in the male reproductive system was being proposed based on scattered data (6, 7) but recent advances have come from in vitro, in vivo and immunohistochemical studies which have begun to elucidate the mechanisms of estrogen action on the male reproductive tract (8-10).
PHYSIOLOGY
Estrogen biosynthesis and actions
In males, estrogens derive from circulating androgens. Aromatization of the C19 androgens, testosterone and androstenedione, to form estradiol and estrone, respectively, is the key step in estrogen biosynthesis, which is under the control of the aromatase enzyme. The aromatase enzyme is a P450 mono-oxygenase enzyme complex present in the smooth endoplasmic reticulum which acts through three consecutive hydroxylation reactions, whose final effect is the aromatization of the A ring of androgens (Figure 1).
Figure 1: Biochemical pathway of testosterone conversion into estrogen in men.
P450 aromatase is the product of the CYP19 gene which consists of at least 16 exons and is located on chromosome 15 in humans (5, 11) (Figure 2).
Figure 2: Schematic representation of the human aromatase gene.
In plasma, estrogens are reversibly bound to sex hormone binding globulin (SHBG), a b-globulin, and, to a lesser degree to albumin. Estrogen actions are mediated by binding to specific nuclear estrogen receptors (ERs), which are ligand-inducible transcription factors regulating the expression of target genes after hormone binding. Two subtypes of ERs have been described: estrogen receptor a (ERa) and the more recently discovered estrogen receptor b (ERb). The human gene encoding for ERa is located on the long arm of chromosome 6, while the gene encoding for ERb is located on band q22-24 of chromosome 14. The two ER (a and b) proteins have a high degree of homology at the amino acid level (Figure 3).
Figure 3: ERs gene and its products.
While it is clear that estrogens regulate transcription via a nuclear interaction after binding their receptors, a non-genomic action of estrogens has been recently demonstrated, suggesting a different molecular mechanism accounts for some estrogen actions. In vitro studies showed a very short latency time between the administration of estrogens and the appearance of biological effects. These actions are thought to be mediated through cell-surface receptors, which are not believed to act via a transcriptional mechanism (12).
Distribution of ERs and aromatase in the male reproductive system
ERs and the aromatase enzyme are widely expressed in the male reproductive tract in both animals and humans, implying that estrogen biosynthesis occurs in the male reproductive tract and that both locally produced and circulating estrogens may interact with ERs in an intracrine/paracrine and/or endocrine fashion (12). The concept of a key estrogen action in the male reproductive tract is strongly supported by the fact that male reproductive structures are able to produce and respond to estrogens (13).
Distribution of ERs and aromatase in the human male reproductive system
Both ERs have been found in human testis and reproductive tract. In the male fetus ERb expression is higher than ERa, the latter being absent or expressed at very low levels. In the human fetus ERb immunoreactivity has been shown in the seminiferous epithelium (Sertoli cells and a few germ cells) and in the epididymis, while ERa was undetectable in these structures, suggesting a role for ERb in the prenatal development and function of male reproductive structures (28).
ERb has been detected in rodent (21) as well as in primate germ cells (20). In adult men ERa was expressed only in Leydig cells, while ERb was found in the efferent ducts. No ERs were detected in the epididymis. Otherwise, ERb has been documented in both Leydig and Sertoli cells (29). Both ERa and b have been detected in human pachytene spermatocytes and round spermatids with in situ hybridization (30, 31). These latter studies have been contradicted by more recent studies showing strong expression of ERb in human testis but failing to find evidence for ERa using immunohistochemistry (32) and RT PCR (33), suggesting that ERb is the primary mediator of estrogen action in the human testis. Of particular interest is the demonstration of differential expression of wild type ERb (ERb1) and a novel human variant form of ERb, arising from alternate splicing (ERbcx, or ERb2), in the human testis (34). ERb2, which may act as a dominant negative inhibitor of ER action, was highest in spermatogonia and Sertoli cells in adult men, suggesting that these cells may be "protected" from estrogen action by the expression of this variant. However wild type ERb1 was highest in pachytene spermatocytes and round spermatids, which have been proposed to be estrogen sensitive (see 13 for review), yet was low in less mature germ cells (32).
As previously suggested by Durkee et al. (35), ERs are present in human sperm. In particular it has recently been documented by Luconi et al (9) that the sperm membrane contains an estrogen receptor-related protein able to bind steroid hormones which may act through a calcium-calmodulin dependent pathway and thus perhaps accounts for a well documented rapid non-genomic action.
Aromatase expression in the human testis is present in both somatic and germ cells from pachytene spermatocytes through elongated spermatids (36, 16, 24, 27). Aromatase is also expressed in both human Leydig and Sertoli cells (24, 36). Recently, the presence of aromatase has been demonstrated not only in immature germ cells (24, 27), but also in mature human spermatozoa (37, 16). In contrast to rodents, aromatase expression in human gametes is not lost during transit through the genital tracts since P450 aromatase was demonstrated in ejaculated human spermatozoa at three different functional levels: mRNA expression (37), protein and activity (8). Thus ejaculated human spermatozoa continue to express P450 aromatase and contain active aromatase, and thus sperm have to be considered a potential site of estrogen biosynthesis.
In summary, the testes are able to synthesize and respond to estrogens throughout development. The localization of ERa, ERb and aromatase suggests that estrogen action is likely to be important for testicular and efferent ductule function. The role of estrogens in the male reproductive system has become clearer in regard to animals, and the mapping of ERs and aromatase distribution in the human male reproductive system has led to the suggestion that estrogen plays a role in human male reproduction. As a consequence, a new field of research has evolved aimed at improving our knowledge of estrogen action on male reproduction and the molecular mechanisms involved in both animals and men. To date some estrogen actions on male reproduction have been well characterized but more research is in progress to further define the nature of estrogen action, as outlined in the following section.
Role of estrogens in male reproduction
Role of estrogens in animal male reproduction
In animals, a previously unsuspected physiological role of estrogens in testicular function was revealed by the creation of the ERa knockout (aERKO) mouse. Adult, sexually mature, male aERKO mice are infertile even though the development of the male reproductive tract is largely unaffected (3). Adult testicular histology shows an atrophic and degenerating seminiferous epithelium, together with dilated tubules and a dilation of the rete testis (38). The disruption of spermatogenesis is progressive as the testicular histology is normal at ten days of age but starts to degenerate at twenty-thirty days. By about 40-60 days the tubules are markedly dilated with a corresponding significant increase in testicular volume while the seminiferous epithelium becomes atrophic (3). A severe impairment in tubule fluid absorption in the efferent ducts was demonstrated to be the cause of infertility in aERKO male mice, and this defect is partially mimicked also by the administration of an anti-estrogen in wild-type mice (19). In the male genital tract the highest concentration of ERa is found in the efferent ducts (39) and the estrogen-dependent fluid reabsorption in this site probably results from estrogen interaction with the ERa. The lack of fluid reabsorption in the efferent ductules of aERKO male mice and the consequent dilatation of these ductules induces a retroactive progressive swelling of the seminiferous tubules. The seminiferous tubule damage results from the increased fluid pressure and severely impaired spermatogenesis coupled with testicular atrophy as seen at the age of 150 days (19, 3). In addition, reproductive hormones profiles are abnormal in aERKO male mice as serum LH is significantly increased with a consequent elevated serum testosterone and Leydig cells hyperplasia, but FSH remains in the normal range (3). It is also worth noting that detailed investigations into the development of efferent ductules in aERKO male mice suggest that a congenital absence of ERa leads to developmental abnormalities in this tissue (40).
The recent production of both aromatase knockout (ArKO, 41) and ERb knockout (bERKO, 42) mice supports the idea that in mice estrogen actions on the male reproductive tract are more complex than previously suggested on the basis of the aERKO mice (3). In fact, unlike aERKO mice, male ArKO mice are initially fully fertile (41), but fertility decreases with advancing age (43), and, conversely, bERKO mice are fully fertile and apparently reproductively normal in adulthood (42).
From seven months of age male ArKO mice are not able to sire any litters. Again histology of the testes of one-year-old ArKO mice shows a disruption of spermatogenesis at the early spermatid without significant changes in the volume of seminiferous tubule lumen, together with Leydig cell hyperplasia (43). Despite the phenotype of aERKO male mice, the mechanism involved in the development of infertility is different in ArKO male mice, since the early arrest of spermatogenesis suggests a failure of germ cell differentiation probably caused by the lack of estrogen action at the level of the seminiferous epithelium rather than a problem referable to impaired fluid reabsorption (9, 15). Recent findings from studies in which human germ cells were treated with estrogen in vitro suggest that estradiol may serve as a survival factor for round spermatids and that lack of estradiol may promote apoptosis with a resulting failure in elongated spermatid differentiation (31). Recently studies in mice deficient in both ER a and b (abERKO mice) showed a male phenotype very close to that of aERKO mice with infertility and dilated seminiferous tubules (3). These findings, together with the observation that bERKO male mice are fully fertile (42), lead to the hypothesis that estrogen activity in the male reproductive tract differs with regard to both the type of estrogen receptor involved in the pathway of estrogenic action and the site of action through the male reproductive tract. Importantly, results from mice lacking functional ERs or aromatase point to an important role for estrogen in the maintenance of mating behaviour in male mice, and that infertility in aERKO, abERKO and ArKO mice are at least in part due to reductions in various aspects of mating behavior from an early age (see 3, 13 for review).
The above studies support the concept that a functional ERa, but not ERb, is needed for the development and maintenance of a normal fertility in male mice (3, 19, 38, 42). Clearly, further studies are needed to fully understand the precise role of estrogens and their receptors in the establishment and maintenance of male fertility, and the importance of intracrine and paracrine pathways for these effects.
Role of estrogens in human male reproduction
The demonstration of abundant ERs in human efferent ducts and aromatase activity in human sperm, speaks in favor of the involvement of estrogens in male reproductive function. On the other hand, data from human subjects with congenital estrogen deficiency have provided conflicting and somewhat confusing results. The only man with estrogen resistance discovered up till now, a human equivalent of the ERKO mouse, had normal testicular volumes and a normal sperm count but with slightly reduced motility (4) (Table 5). The three adult men affected by congenital aromatase deficiency showed a variable degree of impaired spermatogenesis (14). The patient described by Carani et al., showed both a severely reduced sperm count and an impairment of sperm viability with germ cell arrest at the level of primary spermatocytes (5, 14, 44) (Table 5). A more recent patient had complete germ cell arrest on testicular biopsy but a semen analysis was not performed according to patient's religious views (45, 46) (Table 5). Data concerning the patient described by Morishima et al. are lacking since sperm counts were not analyzed (47) (Table 5). It should be remarked that a clear cause-effect relationship between infertility and aromatase deficiency is not demonstrable in the patient studied by Carani et al., since one of his brothers was infertile despite the absence of mutations in the aromatase gene, suggesting an alternate common cause for their infertility. (44).
Recently a new patient with aromatase deficiency has been described to have impaired fertility (48), confirming a possible association between congenital estrogen deficiency and infertility.
Table 5: Reproductive phenotypes of men with congenital estrogen deficiency.
SUBJECTS REPRODUCTIVE HORMONES EXTERNAL GENITALIA SEMEN ANALYSIS
Estrogen resistance (Age: 28 yrs)
(ref 5) Increased serum LH.
Increased serum FSH.
Normal serum testosterone.
Increased serum estradiol. Normal male genitalia.
Volume of each testis:20-25 mL. Sperm count: 25x106 / mL (normal > 20 x 106 / mL).
Viability: 18% (normal > 50%).
Aromatase deficiency (Age: 24 yrs)
(refs 47 and 63) Increased serum LH.
Increased serum FSH.
Increased serum testosterone.
Undetectable serum estradiol. Normal male genitalia.Volume of each testis: 34 mL Not performed.
Aromatase deficiency (Age: 38 yrs)
(refs 44 and 99) Normal-to-raised serum LH.Increased serum FSHNormal serum testosterone.
Undetectable serum estradiol. Normal male genitalia.Volume of each testis: 8mL. Sperm count: 1x 106 / ml (normal > 20 x 106 / mL). Viability: 0% (normal > 50%).
Testis biopsy: germ cell arrest at primary spermatocyte level.
Aromatase deficiency (Age: 28 yrs)
(refs 45 and 46) Normal serum LH. Increased serum FSH. Low-normal serum testosterone.
Undetectable serum estradiol. Cryptorchidism.
Volume of each testis: 10-11 mL. Not performed.
Testis biopsy: complete germ cell arrest.
Aromatase deficiency (Age: 27 yrs)
(ref 48) Normal serum LH
Increased serum FSH
Increased serum testosterone
Undetectable serum estradiol Volume of each testis: 13-14 mL Sperm count: 17.4 x 106 / ml (normal >20 x 106 / mL).
Viability: reduced
The variable degree of fertility impairment in men with congenital deficiency of estrogen action or synthesis deficiency does not permit a firm conclusion about whether these features are a consequence of a lack of estrogen action or are only epiphenomena, even though a possible role of estrogen on human spermatogenesis is suggested by rodent studies. Recently, the administration of aromatase inhibitors to infertile men with an impaired testosterone to estradiol ratio resulted in an improvement of fertility rate (49), although in the absence of a placebo or control group, these findings need to be interpreted with great caution. Clearly our knowledge of a role for estrogen in human male reproduction is far from complete. The exposure to the excess of environmental estrogens has been proposed as a possible cause of impaired fertility. It is difficult to reconcile existing data about effects of both estrogen deficiency and excess on male reproductive function (1, 6, 50, 51). These issues are discussed further below.
Regulation of gonadotropin feedback
The regulation of gonadotropin feedback is an important and well-documented action of estrogen in males. While testosterone has been classically considered the key hormone for the control of gonadotropin feedback in the male (Figure 4), a role for estrogens has become clear from studies performed in normal and GnRH-deficient men. Recently, the discovery of men with congenital estrogen deficiency has also provided further evidence for the relationship between estrogens and gonadotropin secretion in men (5).
Figure 4: Traditional knowledge concerning sex steroids on the control of gonadotropin secretion.
The effects of estrogens on gonadotropin secretion have been investigated in GnRH deficient males whose gonadotropin secretion was normalized by pulsatile GnRH administration. In order to determine the precise role of sex steroids in the hypothalamo-pituitary-testicular axis, two studies were performed in which testosterone alone, testosterone plus testolactone (an aromatase inhibitor), or estradiol were administered (52, 53). When given testosterone alone, these subjects revealed a significant decrease in mean basal LH and FSH levels as well as LH pulse amplitude, demonstrating a direct suppressive effect on the pituitary of testosterone and its metabolites. Mean LH levels and LH frequency were suppressed to a greater extent in normal control subjects during testosterone administration suggesting also a hypothalamic site of action of testosterone in suppressing GnRH secretion. In order to discriminate the impact of testosterone as opposed to its aromatized products, both groups of subjects were administered with testosterone plus testolactone. The addition of the aromatase inhibitor completely prevented the suppression of gonadotropin secretion by testosterone in both normal and GnRH deficient men: in fact the mean LH levels increased significantly in both groups. The increase in mean LH levels was greater in the normal men who received testolactone alone compared to normal men who received testosterone plus testolactone, thus revealing also a direct effect of androgens in normal men.
It is clear that the aromatization of testosterone into estradiol is required for a normal gonadotropin feedback at the pituitary level (52). In fact, when the same experimental model was applied using estradiol administration, mean LH and FSH levels as well as LH pulse amplitude all decreased significantly during estradiol administration (53). This study demonstrates an important direct inhibitory effect of estradiol on gonadotropin secretion in both the GnRH-deficient and normal men.( 52, 53) and supports the concept that at least part of the inhibitory effect on gonadotropin secretion is mediated by the conversion of testosterone to estradiol (52, 53). In contrast it seems that the 5a-reduction of testosterone to DHT does not play an important role in pituitary secretion of gonadotropins (54).
More recently a hypothalamic site of action of estrogens has been demonstrated in men. In order to clarify the role of estrogen on the feedback regulation of gonadotropin secretion at the hypothalamic level, Hayes et al (55) conducted a study involving the administration of the aromatase inhibitor, anastrozole, to men affected by idiopathic hypogonadotropic hypogonadism (IHH), whose gonadotropin secretion had been normalized by long term pulsatile GnRH therapy. They observed that inhibition of estradiol synthesis led to an increase in mean gonadotropin levels in both normal and IHH men, but with a greater increase in the normal subjects suggesting a hypothalamic mode of action. The rise in mean LH levels in the normal subjects was shown to be due to anastrozole causing an increase in LH pulse frequency and amplitude. The authors concluded that estrogen acts at the hypothalamic level to decrease both GnRH pulse frequency and pituitary responsiveness to GnRH (55).
Accordingly, the effects of estrogen on gonadotropin secretion at the pituitary level has recently been demonstrated to operate from early- to mid-puberty (56, 57) into old age in men (58). The administration of an aromatase inhibitor (anastrozole 1 mg daily for 10 weeks) to boys aged 15-22 years (56) resulted in a 50% decrease in serum estradiol concentrations, an increase in testosterone concentrations and an increase in both LH and FSH values during the whole study protocol. These hormonal parameters returned to normal values after discontinuation of anastrozole treatment. Recently, administration of letrozole, another potent aromatase inhibitor, was shown to increase serum LH, frequency of LH pulse amplitude and the response of LH to GnRH administration in boys during early and mid-pubertal phases, indicating that estrogens acts at the pituitary level during early phases of puberty (57). The same mechanism continues to operate during adulthood and also during early senescence. In fact, in fifteen eugonadal men aged 65 years treated with 2 mg anastrozole for 9 weeks, serum FSH and LH levels increased significantly, in spite of an increase in serum testosterone levels (58).
Data suggests that estradiol may modulate GnRH receptors number and function at the pituitary level (59), although no ERs, (at least of the a type,) were found in GnRH secreting neurons in monkeys (60). However ERb is found in GnRH expressing neurons in male and female rats (61). The precise mechanism of estrogen action at both the hypothalamic and pituitary level in men remains unclear (62). It remains to be established whether estrogen receptors are involved at these two sites and/or whether non-genomic estrogen actions play a role in the control of the gonadotropin feedback. Further studies are needed to establish the contribution of both circulating and locally produced estrogens to gonadotropin feedback as well as the target cells involved in estrogen action within the hypothalamus. Nevertheless it is now well established that some androgens need to be converted to estrogens in order to ensure the integrity of the gonadotropin feedback mechanism in men, testosterone itself having a more minor role than previously thought (Figure 5).
Figure 5: Sex steroids control of gonadotropin secretion after recent advances.
Our understanding of the role of estrogens in gonadotropin feedback has been enhanced through studies of men affected with congenital estrogen deficiency. The description of a man lacking a functional ERa (4) revealed a remarkable hormonal pattern consisting of a normal serum testosterone, high estradiol and estrone levels but increased serum FSH and LH concentrations (Table 5). Other important information about the role of estrogens in the human male has come from the discovery of naturally occurring mutations in the aromatase gene. To date five different cases of human male aromatase deficiency have been described, four of these males were discovered to be aromatase deficient during adulthood and one as a child (44-48, 63, 64). The four adult patients had an increase in basal FSH concentrations, while LH showed a more heterogeneous pattern, being elevated only in one subject (47, 63), high to normal in another (44, 65, 66) and normal in the third and fourth (45, 46, 48). Serum testosterone concentrations were also variable being elevated (47, 48, 63, ), normal (44, 65, 66) and low to normal (45, 46) in the four patients respectively. In all four patients estradiol concentrations were undetectable (Table 5). The demonstration of elevated gonadotropin levels in the presence of normal to increased serum testosterone levels in these men further highlights the important role for estrogen in regulating circulating gonadotropins in men.
A detailed study of the effects of different doses of transdermal estradiol on pituitary function in a man with congenital aromatase deficiency demonstrated that estrogens might control not only basal secretion of gonadotropins but also their responsiveness to GnRH administration. In this study, estrogen administration to a male patient with aromatase deficiency resulted in a decrease in both basal and GnRH-stimulated LH, FSH and a-subunit secretion with the response to GnRH administration being dose-dependent (66) (Figure 6). These results have been recently confirmed in the last case of aromatase deficiency described (48). However, a complete normalization of serum FSH during estradiol treatment was not achieved in the presence of physiological levels of circulating estradiol and supraphysiological levels of estrogens were necessary to obtain FSH normalization (44, 65), however this was attributed to the concomitant severe impairment of patient's spermatogenesis.
Figure 6: Basal and stimulated serum LH levels in a man affected with aromatase deficiency: effect of three different dosages of transdermal estradiol. (TE) (66)
Some difficulties remain with interpreting these data from men with congenital estrogen deficiency. For example, in the young patient with congenital aromatase deficiency, no abnormalities were found in either gonadotropin secretion nor in testis size; both testes were descended and the penis was normal (64). The presence of normal levels of gonadotropins raises the possibility that the role of estrogens in the hypothalamo-pituitary-testicular axis only become relevant in a later stage of life than infancy. Thus the control of gonadotropin feedback exerted by sex steroids during early infancy and childhood remains a matter of debate in the human male.
EFFECTS OF EXCESS ESTROGEN OR ESTROGEN DEFICIENCY ON MALE REPRODUCTION
Exposure to excess estrogens in animals
In order to evaluate the effect of estrogen excess on the reproductive tract, several studies have been performed in various animal species treated with diethylstilbestrol, a synthetic estrogenic compound. In male mice, the critical period for Müllerian duct formation is day 13 post-coitus. Prenatal exposure of fetal male mice to DES caused a delay in Müllerian duct formation by approximately two days as well as incomplete Müllerian duct regression with a female-like differentiation of the non-regressed caudal part (67). An increase in the expression of anti-Müllerian-Hormone (AMH) mRNA in male mice fetuses exposed to DES has also been demonstrated. This increase was not accompanied by a regression of the ducts. This data was interpreted to suggest that the asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, led to the persistence of Müllerian duct remnants at birth in male mice. Moreover DES exposure did not impair embryonal genetic development, but increased ERs number, and slightly prolonged the gestation time (cesarean sections were performed to rescue the litter and revealed no difference in size of fetuses from control and DES treated mothers). The timing of DES exposure is crucial to the induction of abnormalities of Müllerian duct development and regression (67).
Many studies in rodents suggest that inappropriate exposure to estrogen in utero and during the neonatal period impairs testicular descent, efferent ductule function, the hypothalamic-pituitary-gonadal axis, and testicular function (see 2 and 13 for review). The latter effect can be a direct consequence of exposure to excess estrogen, as well as a secondary effect due to perturbations in circulating hormones or the ability of the efferent ductules to reabsorb fluid. Some studies show that low dose estrogenic substances given during puberty can actually stimulate the onset of spermatogenesis, likely due to stimulatory effects on FSH (68), highlighting the fact that the effects of excess estrogen on male fertility are often complex. The effects of excess estrogen in the neonatal period can impact upon the testis into adulthood, with permanent changes in testis function and spermatogenesis evident (see 2 and 13 for review).
Aromatase over-expression in rodents
Recently a transgenic line of mice overexpressing aromatase enzyme (AROM+) has been developed (69, 70). These mice show highly elevated serum estradiol concentrations, with a reciprocal decrease in testosterone concentrations. The AROM+ males display several of the changes observed in males perinatally exposed to estrogens, such as undescended testes, testicular interstitial cell hyperplasia, hypoandrogenism, and growth inhibition of accessory sex glands. A disruption of spermatogenesis has also been observed which could be a consequence of multiple factors, including cryptorchidism, abnormal Leydig cell function, hypoandrogenemia or hyperestrogenemia. Estrogens are thought to inhibit Leydig cell development, growth and function, resulting in the suppression of androgen production (see 13 for review). The observation of numerous degenerating germ cells and the absence of spermatids within the seminiferous tubules of AROM+ mice suggest that germ cells development was arrested at the pachytene spermatocyte stage in the cryptorchid testes. Interestingly, the spermatogenic arrest occurred at a stage where P450arom is typically expressed. The spermatogenic arrest found in the AROM+ mice could be explained, at least partially, by the suppression of FSH action. The reduced serum FSH levels in AROM+ males are further evidence of the inhibiting actions of estrogens on FSH secretion in males. No significant differences in the LH concentrations were seen in AROM+ and wild type mice (69, 70).
Exposure to excess estrogens in humans
The clinical use of diethylstilbestrol (DES) by pregnant women in order to prevent miscarriage resulted in an increased incidence of genital malformations in their sons (71). In these individuals the presence of Müllerian ducts remnants was found indicating that fetal exposure to DES may have an effect on sex differentiation in men, as is the case in rodents (67). Moreover a large number of structural and functional abnormalities were found, the most frequent being: epididymal cysts, meatal stenosis, hypospadias, cryptorchidism and microphallus (71). The frequency of abnormalities was dependent on the timing of estrogen exposure: in fact, men who were exposed to DES before 11th week of gestation (i.e. the time of Müllerian ducts formation) had a two fold higher rate of abnormalities than those who were exposed only later (71). This data supports the previously discussed hypothesis that the asynchrony between formation and regression of embryonal reproductive structures is determined by estrogen exposure.
Various reports have demonstrated that semen quality of men exposed to DES in utero is significantly worse than in unexposed controls (72, 73). However, the sperm concentrations of most of the DES exposed men were well above the limit at which subfertility occurs, and it is therefore not surprising that the fertility of these men was reported to be normal (7). The risk of testicular cancer among men exposed to DES in utero has been a controversial issue and several meta-analyses showed no increased risk (74). However more direct evidence will be necessary in order to fully understand this issue.
While various studies suggest that environmental estrogens affect male fertility in animal models, the implications for human spermatogenesis are less clear (75). It has been demonstrated that male mice whose mothers have consumed a 29 ng/g dose of bisphenol A for seven days during pregnancy had a 20% lower sperm production as compared to control males (76). Various abnormalities in reproductive organs have also been described in males exposed to bisphenols (i.e. a significant decrease in the size of the epididymis and seminal vesicles and an increase in prostate gland volume), suggesting that bisphenols interfere with the normal development of the Wolffian ducts in a dose-related fashion. Exogenous estrogens could interfere with the development of the genital structures if administered during early organogenesis, by leading to both an impairment of gonadotropin secretion and by creating an imbalance in the androgen to estrogen ratio, which may account for impaired androgen receptor stimulation or inhibition according to the dose, the cell type and age (71, 77, 78, 79).
An excess of environmental estrogens has been suggested as a possible cause of impaired fertility in humans (50, 51). A progressive decline in sperm count has been reported in some Western countries during the past 50 years, suggesting a possible negative effect of environmental contaminants on male reproductive function (6, 51, 71, 77). Data concerning the role of estrogens in male reproductive structure development remains conflicting. Animal studies suggest that exposure to estrogen excess may negatively affect the development of reproductive male organs. These effects, however, are considered to be the result of an impaired hypothalamic-pituitary function as a consequence of estrogen excess and of the concomitant androgen deficiency (78, 79). Much of the knowledge on excess estrogen exposure and human fertility depends upon animal data and the validity of these concepts to humans has not been established.
Aromatase over-expression in humans
In 1996 a boy with aromatase excess syndrome was reported (80). His condition was presumably inherited in an autosomal dominant fashion with sex-limited expression as his father had a history of peripubertal gynecomastia, elevated serum estrogen levels and increased aromatase activity in vitro. The father was fertile and had a normal libido despite a small testicular volume (15 mL bilaterally), and a reduced testosterone level of 234 ng/dL(80). In the son, mild suppression of testicular growth and Leydig cell function probably reflected direct estrogen negative feedback on pituitary gonadotropin secretion. In general, the inhibitory effects of estrogen on reproductive function appear to be milder in males with aromatase excess syndrome than in patients receiving exogenous estrogens or with estrogen-secreting tumors, probably because serum estradiol and/or estrone levels are lower in the former (80).
Estrogen deficiency: human models
Effect of estrogen deficiency on gonadotropin feedback
The role of estrogens in human male physiology has become better understood as a result of the description of a man lacking a functional estrogen receptor a (4). This patient presented with tall stature, continuing linear growth during adulthood, unfused epiphyses and osteoporosis. He had normal serum testosterone, high estradiol and estrone levels, and increased FSH and LH concentrations. He had normal male genitalia with bilaterally descended testes each of 20-25 mL volume and a normal prostate volume. No further studies were performed on the reproductive system of this male (Table 5). The only data available are those on sperm characteristics, which revealed a reduced viability of sperm (Table 5).
Other important information about the role of estrogens in the human male came from the discovery of naturally occurring mutations in the aromatase gene. To date only five different cases of human male aromatase deficiency have been found, three of these males were discovered to be aromatase deficient during adulthood and one of them as a child (44-48, 63, 64).
The hormonal pattern of the four adult patients affected by aromatase deficiency is summarized in Table 5. The study of men with aromatase deficiency shows that estrogens modulate gonadotropin feedback by regulating both basal secretion of gonadotropins and their responsiveness to GnRH ( 44, 48, 63, 65, 66).
In the young patient with congenital aromatase deficiency no alterations were found either in gonadotropin secretion or in testis size, both testes were descended and the penis was normal (64). The presence of normal levels of gonadotropins raises the possibility that the role of estrogens in the regulation of the hypothalamo-pituitary-testicular axis becomes relevant in a later stage of life than infancy. This is in contrast to aromatase deficient female patients in whom an elevation of FSH and LH is seen even in childhood (85), demonstrating the importance of estrogens in the feedback regulation of gonadotropin secretion in girls during every stage of life.
Effect of estrogen deficiency on the human testis
Two of the adult males affected by aromatase deficiency showed a decreased testicular volume, one had normal testes, while the other had large testes. The histological study performed in only two of the four patients showed profound alterations in germ cell development, in particular one of the two patients had germ cells arrest at primary spermatocytes and the other had complete depletion of germ cells. Sperm analysis of one of these two patients showed severe oligozoospermia and astenozoospermia (Table 5). It remains unclear as to whether their disordered estrogen physiology accounts for the spermatogenic defects.
Effect of estrogen deficiency on the development of reproductive structures
Bilateral cryptorchidism was present in one patient with aromatase deficiency (45, 46), suggesting a possible role of estrogen also in testis descent, although this was not seen in the transgenic mice models (see above). The presence of a unique case of cryptorchidism among men with aromatase deficiency does not permit any conclusions to be drawn concerning a possible relationship between estrogen deficiency and the occurrence of abnormalities in testis development and descent.
ESTROGEN AND MALE SEXUAL BEHAVIOR
Gender-identity and sexual orientation
Sex steroids and particularly testosterone are able to affect adult male sexual behavior in mammals (86). In non-primate mammals, androgen exposure during late fetal and early neonatal development in the male accounts for the sexual dimorphism of the central nervous system (CNS), probably as a result of testosterone aromatization in the brain (87-92). Prenatal and perinatal estrogen action in the brain is believed to be responsible for the establishment of a male brain (93). Paradoxically, male rat brain is exposed to a greater amount of estradiol than female brain, since ovaries release less estrogen than testes at this stage of development (obviously in male estrogens derives from the conversion of testosterone produced by testis). Furthermore, estrogens are inactivated in the female fetus by various biochemical mechanisms, such as binding to alpha-fetoprotein (94). As a consequence, a sexual dimorphism of hypothalamic structures develops in rodents and the same mechanism seems to be involved also for the establishment of differences in hypothalamic structures between men and women (92, 95-97).
The role of sex steroids and of testosterone aromatization in the determination of the imprinting of sexual behavior has been considered of primary importance for the determination of both adult sexual orientation and sexual behavior in both animals and humans (92, 96-98). Particularly, it was thought that testosterone deficiency and the lack of its estrogenic metabolites during early phases of development could affect sexual orientation (92, 95, 96). Recently, a detailed study of a man with aromatase deficiency did not reveal any abnormalities of both gender-identity and sexual orientation (99). Based on this study the patient was categorized as masculine, his gender identity was male and the psychosexual orientation was heterosexual. Also data from the other men with aromatase deficiency don't show any association between congenital aromatase deficiency and gender-identity or sexual orientation disturbances (4, 45-48) (Table 7). Since aromatase deficient patients would be subjected to maternal estrogens in utero, it is also possible that such estrogen exposure would be sufficient for sexual behavior development.
These data suggest that congenital aromatase deficiency does not affect psychosexual orientation and gender-identity in humans and that, in contrast to animals, human psychological and social factors may be the most relevant determinants of gender role behavior in men, with hormones having a minor role. Hormones, in fact, may affect sexual differentiation and sex assignment at birth and, only indirectly, psychosexual development in men (79).
Sexual behavior
In mammals, adult male sexual behavior is at least partially dependent on the presence of testosterone. Androgens are also necessary for male sexual behavior during adult life (100-102). In fact, the lack of testosterone frequently produces loss of libido and erectile dysfunction (101, 102). At the same time, testosterone replacement therapy increases sexual interest and improves sexual behavior (86, 101). By contrast, the role of aromatization in the establishment and maintenance of male sexual behavior has been characterized only recently.
Congenital aromatase deficiency and estrogen action blockade result in a severe impairment of sexual behavior in rodents. ArKO mice (41) exhibit a significant reduction in mounting frequency and a significantly prolonged latency to mount when compared with heterozygous and wild-type animals (103). Also the sexual behavior of aERKO mice is characterized by a reduction of intromissions, an increase in the latency to first intromission and a lack of ejaculation, despite the presence of a normal motivation to mount females. The same sexual behavior pattern occurs in abERKO male mice (see 3, 13 for review). On the contrary, bERKO mice showed all three components of sexual behavior including ejaculation (Figure 7). These findings suggest that at least one of the ERs (ERa) is required for the expression of simple mounting behavior in male mice and, as a consequence, that activation of the androgen receptor alone is not sufficient for a fully normal sexual behavior, confirming that aromatization of androgens is also required.
Figure 7: Sexual behavior in estrogen deficient male mice.
The role of estrogens in male sexual behavior is confirmed by studies in gonadectomized rats treated with testosterone (104). Vagell and McGinnis showed, in fact, a complete inhibition of male sexual behavior in gonadectomized rats when the aromatase inhibitor fadrozole was administered in addition to testosterone, demonstrating that this inhibition disappeared when estrogen administration was added (104).
Much less is known about the role of estrogens in sexual behavior in the human male, particularly the degree to which the effects of testosterone ought really be ascribed to its conversion into estradiol. Previous studies aimed at addressing this issue have provided conflicting results both in support (105, 106) and against (107, 108) an important role of estrogen on human male sexuality. In order to evaluate the role of estrogens in human male sexual behavior, sexual activity has been investigated in a man with aromatase deficiency, before and during testosterone or transdermal estradiol treatment. When the patient received his physiological dose of estrogens (i.e. 25 mg transdermal estradiol twice weekly) he experienced an increase of all the parameters of sexual activity (the frequency of masturbation, sexual intercourse, erotic fantasies and libido) (Table 7, Figure 8), without change during testosterone treatment.
In men with congenital estrogen deficiency it seems that estrogen may play a role in adult sexual behavior, even if it's not possible to exclude that the improvements observed were the result of an improvement in well being and mood related to the estrogen replacement therapy.
Figure 8: Sexual behavior parameters in a men with aromatase deficiency:
effect of testosterone and estradiol treatment.
PHASE A: before testosterone treatment.
PHASE B: during testosterone treatment (testosterone enanthate): 250 mg every 10 days i.m for 6 months.
PHASE C: before estradiol treatment.
PHASE D: during transdermal estradiol treatment: 50 mg twice/week transdermal estradiol for 6 months.
PHASE E: during transdermal estradiol treatment: 25 mg twice/week transdermal estradiol for 9 months.
PHASE F: during transdermal estradiol treatment: 12,5 mg twice/week transdermal estradiol for 9 months.
These findings from transgenic mice and humans deficient in aromatase suggest that physiological levels of estrogens could be required for completely normal sexual behavior .
CONCLUSIONS
Sex steroids account for sexual dimorphism because they are responsible for the establishment of primary and secondary sexual characteristics, which are under the control of androgens and estrogens in male and female, respectively. (Figure 9).
Figure 9: Direct and indirect (estrogen-mediated) testosterone action.
Advances in the understanding of the role of estrogens in animal and human models suggest a role for this sex steroid in the reproductive function of both sexes. The fact that both estrogen excess and estrogen deficiency influence male sexual development, testis function, the hypothalamic-pituitary-testis axis, spermatogenesis and ultimately male fertility highlight the importance of estrogen action in the male. From an evolutionary perspective this provides an example of the parsimony operating in biological events which are crucial for the evolution of the human species such as growth and reproduction.
This chapter has been concerned with the reproductive effects of estrogens in males but there are emerging roles for estrogens in non-reproductive tissues. In particular, while traditionally, testosterone has been considered the sex hormone involved in bone maturation and growth arrest in men, but recently the key role of estrogens on growth has been emphasised (5, 44). In men and women, in fact, epiphyseal closure and growth arrest are not achieved without estrogens, underlining the fact that estrogens on human growth are highly conserved in both sexes. Thus it is clear that testosterone might act directly or through its conversion into estrogens (109). This aspect of estogen action is discussed in Chapter 2 and Chapter 3 in this section.
Go back to ENDOCRINOLOGY OF MALE REPRODUCTION
ESTROGENS AND MALE REPRODUCTION
Vincenzo Rochira, Fabbi Matteo, Valassi Elena and Cesare Carani
January 7, 2003 Inde
INTRODUCTION
The intriguing concept that a role for estrogens exists in male reproduction has recently been recognized in the field of endocrinology (1, 2). It has developed from observations and studies that have been performed over the last 10 years. In particular, the development of lines of male transgenic mice lacking functional estrogen receptors or a functional aromatase enzyme have shed new light on the role for estrogens in male reproduction (3). Concomitantly, the discovery of mutations in both the human estrogen receptor alpha (4) and aromatase (5) genes have reinforced the idea that estrogens play a key role in the human male reproductive system.
Previously a role for estrogen action in the male reproductive system was being proposed based on scattered data (6, 7) but recent advances have come from in vitro, in vivo and immunohistochemical studies which have begun to elucidate the mechanisms of estrogen action on the male reproductive tract (8-10).
PHYSIOLOGY
Estrogen biosynthesis and actions
In males, estrogens derive from circulating androgens. Aromatization of the C19 androgens, testosterone and androstenedione, to form estradiol and estrone, respectively, is the key step in estrogen biosynthesis, which is under the control of the aromatase enzyme. The aromatase enzyme is a P450 mono-oxygenase enzyme complex present in the smooth endoplasmic reticulum which acts through three consecutive hydroxylation reactions, whose final effect is the aromatization of the A ring of androgens (Figure 1).
Figure 1: Biochemical pathway of testosterone conversion into estrogen in men.
P450 aromatase is the product of the CYP19 gene which consists of at least 16 exons and is located on chromosome 15 in humans (5, 11) (Figure 2).
Figure 2: Schematic representation of the human aromatase gene.
In plasma, estrogens are reversibly bound to sex hormone binding globulin (SHBG), a b-globulin, and, to a lesser degree to albumin. Estrogen actions are mediated by binding to specific nuclear estrogen receptors (ERs), which are ligand-inducible transcription factors regulating the expression of target genes after hormone binding. Two subtypes of ERs have been described: estrogen receptor a (ERa) and the more recently discovered estrogen receptor b (ERb). The human gene encoding for ERa is located on the long arm of chromosome 6, while the gene encoding for ERb is located on band q22-24 of chromosome 14. The two ER (a and b) proteins have a high degree of homology at the amino acid level (Figure 3).
Figure 3: ERs gene and its products.
While it is clear that estrogens regulate transcription via a nuclear interaction after binding their receptors, a non-genomic action of estrogens has been recently demonstrated, suggesting a different molecular mechanism accounts for some estrogen actions. In vitro studies showed a very short latency time between the administration of estrogens and the appearance of biological effects. These actions are thought to be mediated through cell-surface receptors, which are not believed to act via a transcriptional mechanism (12).
Distribution of ERs and aromatase in the male reproductive system
ERs and the aromatase enzyme are widely expressed in the male reproductive tract in both animals and humans, implying that estrogen biosynthesis occurs in the male reproductive tract and that both locally produced and circulating estrogens may interact with ERs in an intracrine/paracrine and/or endocrine fashion (12). The concept of a key estrogen action in the male reproductive tract is strongly supported by the fact that male reproductive structures are able to produce and respond to estrogens (13).
Distribution of ERs and aromatase in the human male reproductive system
Both ERs have been found in human testis and reproductive tract. In the male fetus ERb expression is higher than ERa, the latter being absent or expressed at very low levels. In the human fetus ERb immunoreactivity has been shown in the seminiferous epithelium (Sertoli cells and a few germ cells) and in the epididymis, while ERa was undetectable in these structures, suggesting a role for ERb in the prenatal development and function of male reproductive structures (28).
ERb has been detected in rodent (21) as well as in primate germ cells (20). In adult men ERa was expressed only in Leydig cells, while ERb was found in the efferent ducts. No ERs were detected in the epididymis. Otherwise, ERb has been documented in both Leydig and Sertoli cells (29). Both ERa and b have been detected in human pachytene spermatocytes and round spermatids with in situ hybridization (30, 31). These latter studies have been contradicted by more recent studies showing strong expression of ERb in human testis but failing to find evidence for ERa using immunohistochemistry (32) and RT PCR (33), suggesting that ERb is the primary mediator of estrogen action in the human testis. Of particular interest is the demonstration of differential expression of wild type ERb (ERb1) and a novel human variant form of ERb, arising from alternate splicing (ERbcx, or ERb2), in the human testis (34). ERb2, which may act as a dominant negative inhibitor of ER action, was highest in spermatogonia and Sertoli cells in adult men, suggesting that these cells may be "protected" from estrogen action by the expression of this variant. However wild type ERb1 was highest in pachytene spermatocytes and round spermatids, which have been proposed to be estrogen sensitive (see 13 for review), yet was low in less mature germ cells (32).
As previously suggested by Durkee et al. (35), ERs are present in human sperm. In particular it has recently been documented by Luconi et al (9) that the sperm membrane contains an estrogen receptor-related protein able to bind steroid hormones which may act through a calcium-calmodulin dependent pathway and thus perhaps accounts for a well documented rapid non-genomic action.
Aromatase expression in the human testis is present in both somatic and germ cells from pachytene spermatocytes through elongated spermatids (36, 16, 24, 27). Aromatase is also expressed in both human Leydig and Sertoli cells (24, 36). Recently, the presence of aromatase has been demonstrated not only in immature germ cells (24, 27), but also in mature human spermatozoa (37, 16). In contrast to rodents, aromatase expression in human gametes is not lost during transit through the genital tracts since P450 aromatase was demonstrated in ejaculated human spermatozoa at three different functional levels: mRNA expression (37), protein and activity (8). Thus ejaculated human spermatozoa continue to express P450 aromatase and contain active aromatase, and thus sperm have to be considered a potential site of estrogen biosynthesis.
In summary, the testes are able to synthesize and respond to estrogens throughout development. The localization of ERa, ERb and aromatase suggests that estrogen action is likely to be important for testicular and efferent ductule function. The role of estrogens in the male reproductive system has become clearer in regard to animals, and the mapping of ERs and aromatase distribution in the human male reproductive system has led to the suggestion that estrogen plays a role in human male reproduction. As a consequence, a new field of research has evolved aimed at improving our knowledge of estrogen action on male reproduction and the molecular mechanisms involved in both animals and men. To date some estrogen actions on male reproduction have been well characterized but more research is in progress to further define the nature of estrogen action, as outlined in the following section.
Role of estrogens in male reproduction
Role of estrogens in animal male reproduction
In animals, a previously unsuspected physiological role of estrogens in testicular function was revealed by the creation of the ERa knockout (aERKO) mouse. Adult, sexually mature, male aERKO mice are infertile even though the development of the male reproductive tract is largely unaffected (3). Adult testicular histology shows an atrophic and degenerating seminiferous epithelium, together with dilated tubules and a dilation of the rete testis (38). The disruption of spermatogenesis is progressive as the testicular histology is normal at ten days of age but starts to degenerate at twenty-thirty days. By about 40-60 days the tubules are markedly dilated with a corresponding significant increase in testicular volume while the seminiferous epithelium becomes atrophic (3). A severe impairment in tubule fluid absorption in the efferent ducts was demonstrated to be the cause of infertility in aERKO male mice, and this defect is partially mimicked also by the administration of an anti-estrogen in wild-type mice (19). In the male genital tract the highest concentration of ERa is found in the efferent ducts (39) and the estrogen-dependent fluid reabsorption in this site probably results from estrogen interaction with the ERa. The lack of fluid reabsorption in the efferent ductules of aERKO male mice and the consequent dilatation of these ductules induces a retroactive progressive swelling of the seminiferous tubules. The seminiferous tubule damage results from the increased fluid pressure and severely impaired spermatogenesis coupled with testicular atrophy as seen at the age of 150 days (19, 3). In addition, reproductive hormones profiles are abnormal in aERKO male mice as serum LH is significantly increased with a consequent elevated serum testosterone and Leydig cells hyperplasia, but FSH remains in the normal range (3). It is also worth noting that detailed investigations into the development of efferent ductules in aERKO male mice suggest that a congenital absence of ERa leads to developmental abnormalities in this tissue (40).
The recent production of both aromatase knockout (ArKO, 41) and ERb knockout (bERKO, 42) mice supports the idea that in mice estrogen actions on the male reproductive tract are more complex than previously suggested on the basis of the aERKO mice (3). In fact, unlike aERKO mice, male ArKO mice are initially fully fertile (41), but fertility decreases with advancing age (43), and, conversely, bERKO mice are fully fertile and apparently reproductively normal in adulthood (42).
From seven months of age male ArKO mice are not able to sire any litters. Again histology of the testes of one-year-old ArKO mice shows a disruption of spermatogenesis at the early spermatid without significant changes in the volume of seminiferous tubule lumen, together with Leydig cell hyperplasia (43). Despite the phenotype of aERKO male mice, the mechanism involved in the development of infertility is different in ArKO male mice, since the early arrest of spermatogenesis suggests a failure of germ cell differentiation probably caused by the lack of estrogen action at the level of the seminiferous epithelium rather than a problem referable to impaired fluid reabsorption (9, 15). Recent findings from studies in which human germ cells were treated with estrogen in vitro suggest that estradiol may serve as a survival factor for round spermatids and that lack of estradiol may promote apoptosis with a resulting failure in elongated spermatid differentiation (31). Recently studies in mice deficient in both ER a and b (abERKO mice) showed a male phenotype very close to that of aERKO mice with infertility and dilated seminiferous tubules (3). These findings, together with the observation that bERKO male mice are fully fertile (42), lead to the hypothesis that estrogen activity in the male reproductive tract differs with regard to both the type of estrogen receptor involved in the pathway of estrogenic action and the site of action through the male reproductive tract. Importantly, results from mice lacking functional ERs or aromatase point to an important role for estrogen in the maintenance of mating behaviour in male mice, and that infertility in aERKO, abERKO and ArKO mice are at least in part due to reductions in various aspects of mating behavior from an early age (see 3, 13 for review).
The above studies support the concept that a functional ERa, but not ERb, is needed for the development and maintenance of a normal fertility in male mice (3, 19, 38, 42). Clearly, further studies are needed to fully understand the precise role of estrogens and their receptors in the establishment and maintenance of male fertility, and the importance of intracrine and paracrine pathways for these effects.
Role of estrogens in human male reproduction
The demonstration of abundant ERs in human efferent ducts and aromatase activity in human sperm, speaks in favor of the involvement of estrogens in male reproductive function. On the other hand, data from human subjects with congenital estrogen deficiency have provided conflicting and somewhat confusing results. The only man with estrogen resistance discovered up till now, a human equivalent of the ERKO mouse, had normal testicular volumes and a normal sperm count but with slightly reduced motility (4) (Table 5). The three adult men affected by congenital aromatase deficiency showed a variable degree of impaired spermatogenesis (14). The patient described by Carani et al., showed both a severely reduced sperm count and an impairment of sperm viability with germ cell arrest at the level of primary spermatocytes (5, 14, 44) (Table 5). A more recent patient had complete germ cell arrest on testicular biopsy but a semen analysis was not performed according to patient's religious views (45, 46) (Table 5). Data concerning the patient described by Morishima et al. are lacking since sperm counts were not analyzed (47) (Table 5). It should be remarked that a clear cause-effect relationship between infertility and aromatase deficiency is not demonstrable in the patient studied by Carani et al., since one of his brothers was infertile despite the absence of mutations in the aromatase gene, suggesting an alternate common cause for their infertility. (44).
Recently a new patient with aromatase deficiency has been described to have impaired fertility (48), confirming a possible association between congenital estrogen deficiency and infertility.
Table 5: Reproductive phenotypes of men with congenital estrogen deficiency.
SUBJECTS REPRODUCTIVE HORMONES EXTERNAL GENITALIA SEMEN ANALYSIS
Estrogen resistance (Age: 28 yrs)
(ref 5) Increased serum LH.
Increased serum FSH.
Normal serum testosterone.
Increased serum estradiol. Normal male genitalia.
Volume of each testis:20-25 mL. Sperm count: 25x106 / mL (normal > 20 x 106 / mL).
Viability: 18% (normal > 50%).
Aromatase deficiency (Age: 24 yrs)
(refs 47 and 63) Increased serum LH.
Increased serum FSH.
Increased serum testosterone.
Undetectable serum estradiol. Normal male genitalia.Volume of each testis: 34 mL Not performed.
Aromatase deficiency (Age: 38 yrs)
(refs 44 and 99) Normal-to-raised serum LH.Increased serum FSHNormal serum testosterone.
Undetectable serum estradiol. Normal male genitalia.Volume of each testis: 8mL. Sperm count: 1x 106 / ml (normal > 20 x 106 / mL). Viability: 0% (normal > 50%).
Testis biopsy: germ cell arrest at primary spermatocyte level.
Aromatase deficiency (Age: 28 yrs)
(refs 45 and 46) Normal serum LH. Increased serum FSH. Low-normal serum testosterone.
Undetectable serum estradiol. Cryptorchidism.
Volume of each testis: 10-11 mL. Not performed.
Testis biopsy: complete germ cell arrest.
Aromatase deficiency (Age: 27 yrs)
(ref 48) Normal serum LH
Increased serum FSH
Increased serum testosterone
Undetectable serum estradiol Volume of each testis: 13-14 mL Sperm count: 17.4 x 106 / ml (normal >20 x 106 / mL).
Viability: reduced
The variable degree of fertility impairment in men with congenital deficiency of estrogen action or synthesis deficiency does not permit a firm conclusion about whether these features are a consequence of a lack of estrogen action or are only epiphenomena, even though a possible role of estrogen on human spermatogenesis is suggested by rodent studies. Recently, the administration of aromatase inhibitors to infertile men with an impaired testosterone to estradiol ratio resulted in an improvement of fertility rate (49), although in the absence of a placebo or control group, these findings need to be interpreted with great caution. Clearly our knowledge of a role for estrogen in human male reproduction is far from complete. The exposure to the excess of environmental estrogens has been proposed as a possible cause of impaired fertility. It is difficult to reconcile existing data about effects of both estrogen deficiency and excess on male reproductive function (1, 6, 50, 51). These issues are discussed further below.
Regulation of gonadotropin feedback
The regulation of gonadotropin feedback is an important and well-documented action of estrogen in males. While testosterone has been classically considered the key hormone for the control of gonadotropin feedback in the male (Figure 4), a role for estrogens has become clear from studies performed in normal and GnRH-deficient men. Recently, the discovery of men with congenital estrogen deficiency has also provided further evidence for the relationship between estrogens and gonadotropin secretion in men (5).
Figure 4: Traditional knowledge concerning sex steroids on the control of gonadotropin secretion.
The effects of estrogens on gonadotropin secretion have been investigated in GnRH deficient males whose gonadotropin secretion was normalized by pulsatile GnRH administration. In order to determine the precise role of sex steroids in the hypothalamo-pituitary-testicular axis, two studies were performed in which testosterone alone, testosterone plus testolactone (an aromatase inhibitor), or estradiol were administered (52, 53). When given testosterone alone, these subjects revealed a significant decrease in mean basal LH and FSH levels as well as LH pulse amplitude, demonstrating a direct suppressive effect on the pituitary of testosterone and its metabolites. Mean LH levels and LH frequency were suppressed to a greater extent in normal control subjects during testosterone administration suggesting also a hypothalamic site of action of testosterone in suppressing GnRH secretion. In order to discriminate the impact of testosterone as opposed to its aromatized products, both groups of subjects were administered with testosterone plus testolactone. The addition of the aromatase inhibitor completely prevented the suppression of gonadotropin secretion by testosterone in both normal and GnRH deficient men: in fact the mean LH levels increased significantly in both groups. The increase in mean LH levels was greater in the normal men who received testolactone alone compared to normal men who received testosterone plus testolactone, thus revealing also a direct effect of androgens in normal men.
It is clear that the aromatization of testosterone into estradiol is required for a normal gonadotropin feedback at the pituitary level (52). In fact, when the same experimental model was applied using estradiol administration, mean LH and FSH levels as well as LH pulse amplitude all decreased significantly during estradiol administration (53). This study demonstrates an important direct inhibitory effect of estradiol on gonadotropin secretion in both the GnRH-deficient and normal men.( 52, 53) and supports the concept that at least part of the inhibitory effect on gonadotropin secretion is mediated by the conversion of testosterone to estradiol (52, 53). In contrast it seems that the 5a-reduction of testosterone to DHT does not play an important role in pituitary secretion of gonadotropins (54).
More recently a hypothalamic site of action of estrogens has been demonstrated in men. In order to clarify the role of estrogen on the feedback regulation of gonadotropin secretion at the hypothalamic level, Hayes et al (55) conducted a study involving the administration of the aromatase inhibitor, anastrozole, to men affected by idiopathic hypogonadotropic hypogonadism (IHH), whose gonadotropin secretion had been normalized by long term pulsatile GnRH therapy. They observed that inhibition of estradiol synthesis led to an increase in mean gonadotropin levels in both normal and IHH men, but with a greater increase in the normal subjects suggesting a hypothalamic mode of action. The rise in mean LH levels in the normal subjects was shown to be due to anastrozole causing an increase in LH pulse frequency and amplitude. The authors concluded that estrogen acts at the hypothalamic level to decrease both GnRH pulse frequency and pituitary responsiveness to GnRH (55).
Accordingly, the effects of estrogen on gonadotropin secretion at the pituitary level has recently been demonstrated to operate from early- to mid-puberty (56, 57) into old age in men (58). The administration of an aromatase inhibitor (anastrozole 1 mg daily for 10 weeks) to boys aged 15-22 years (56) resulted in a 50% decrease in serum estradiol concentrations, an increase in testosterone concentrations and an increase in both LH and FSH values during the whole study protocol. These hormonal parameters returned to normal values after discontinuation of anastrozole treatment. Recently, administration of letrozole, another potent aromatase inhibitor, was shown to increase serum LH, frequency of LH pulse amplitude and the response of LH to GnRH administration in boys during early and mid-pubertal phases, indicating that estrogens acts at the pituitary level during early phases of puberty (57). The same mechanism continues to operate during adulthood and also during early senescence. In fact, in fifteen eugonadal men aged 65 years treated with 2 mg anastrozole for 9 weeks, serum FSH and LH levels increased significantly, in spite of an increase in serum testosterone levels (58).
Data suggests that estradiol may modulate GnRH receptors number and function at the pituitary level (59), although no ERs, (at least of the a type,) were found in GnRH secreting neurons in monkeys (60). However ERb is found in GnRH expressing neurons in male and female rats (61). The precise mechanism of estrogen action at both the hypothalamic and pituitary level in men remains unclear (62). It remains to be established whether estrogen receptors are involved at these two sites and/or whether non-genomic estrogen actions play a role in the control of the gonadotropin feedback. Further studies are needed to establish the contribution of both circulating and locally produced estrogens to gonadotropin feedback as well as the target cells involved in estrogen action within the hypothalamus. Nevertheless it is now well established that some androgens need to be converted to estrogens in order to ensure the integrity of the gonadotropin feedback mechanism in men, testosterone itself having a more minor role than previously thought (Figure 5).
Figure 5: Sex steroids control of gonadotropin secretion after recent advances.
Our understanding of the role of estrogens in gonadotropin feedback has been enhanced through studies of men affected with congenital estrogen deficiency. The description of a man lacking a functional ERa (4) revealed a remarkable hormonal pattern consisting of a normal serum testosterone, high estradiol and estrone levels but increased serum FSH and LH concentrations (Table 5). Other important information about the role of estrogens in the human male has come from the discovery of naturally occurring mutations in the aromatase gene. To date five different cases of human male aromatase deficiency have been described, four of these males were discovered to be aromatase deficient during adulthood and one as a child (44-48, 63, 64). The four adult patients had an increase in basal FSH concentrations, while LH showed a more heterogeneous pattern, being elevated only in one subject (47, 63), high to normal in another (44, 65, 66) and normal in the third and fourth (45, 46, 48). Serum testosterone concentrations were also variable being elevated (47, 48, 63, ), normal (44, 65, 66) and low to normal (45, 46) in the four patients respectively. In all four patients estradiol concentrations were undetectable (Table 5). The demonstration of elevated gonadotropin levels in the presence of normal to increased serum testosterone levels in these men further highlights the important role for estrogen in regulating circulating gonadotropins in men.
A detailed study of the effects of different doses of transdermal estradiol on pituitary function in a man with congenital aromatase deficiency demonstrated that estrogens might control not only basal secretion of gonadotropins but also their responsiveness to GnRH administration. In this study, estrogen administration to a male patient with aromatase deficiency resulted in a decrease in both basal and GnRH-stimulated LH, FSH and a-subunit secretion with the response to GnRH administration being dose-dependent (66) (Figure 6). These results have been recently confirmed in the last case of aromatase deficiency described (48). However, a complete normalization of serum FSH during estradiol treatment was not achieved in the presence of physiological levels of circulating estradiol and supraphysiological levels of estrogens were necessary to obtain FSH normalization (44, 65), however this was attributed to the concomitant severe impairment of patient's spermatogenesis.
Figure 6: Basal and stimulated serum LH levels in a man affected with aromatase deficiency: effect of three different dosages of transdermal estradiol. (TE) (66)
Some difficulties remain with interpreting these data from men with congenital estrogen deficiency. For example, in the young patient with congenital aromatase deficiency, no abnormalities were found in either gonadotropin secretion nor in testis size; both testes were descended and the penis was normal (64). The presence of normal levels of gonadotropins raises the possibility that the role of estrogens in the hypothalamo-pituitary-testicular axis only become relevant in a later stage of life than infancy. Thus the control of gonadotropin feedback exerted by sex steroids during early infancy and childhood remains a matter of debate in the human male.
EFFECTS OF EXCESS ESTROGEN OR ESTROGEN DEFICIENCY ON MALE REPRODUCTION
Exposure to excess estrogens in animals
In order to evaluate the effect of estrogen excess on the reproductive tract, several studies have been performed in various animal species treated with diethylstilbestrol, a synthetic estrogenic compound. In male mice, the critical period for Müllerian duct formation is day 13 post-coitus. Prenatal exposure of fetal male mice to DES caused a delay in Müllerian duct formation by approximately two days as well as incomplete Müllerian duct regression with a female-like differentiation of the non-regressed caudal part (67). An increase in the expression of anti-Müllerian-Hormone (AMH) mRNA in male mice fetuses exposed to DES has also been demonstrated. This increase was not accompanied by a regression of the ducts. This data was interpreted to suggest that the asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, led to the persistence of Müllerian duct remnants at birth in male mice. Moreover DES exposure did not impair embryonal genetic development, but increased ERs number, and slightly prolonged the gestation time (cesarean sections were performed to rescue the litter and revealed no difference in size of fetuses from control and DES treated mothers). The timing of DES exposure is crucial to the induction of abnormalities of Müllerian duct development and regression (67).
Many studies in rodents suggest that inappropriate exposure to estrogen in utero and during the neonatal period impairs testicular descent, efferent ductule function, the hypothalamic-pituitary-gonadal axis, and testicular function (see 2 and 13 for review). The latter effect can be a direct consequence of exposure to excess estrogen, as well as a secondary effect due to perturbations in circulating hormones or the ability of the efferent ductules to reabsorb fluid. Some studies show that low dose estrogenic substances given during puberty can actually stimulate the onset of spermatogenesis, likely due to stimulatory effects on FSH (68), highlighting the fact that the effects of excess estrogen on male fertility are often complex. The effects of excess estrogen in the neonatal period can impact upon the testis into adulthood, with permanent changes in testis function and spermatogenesis evident (see 2 and 13 for review).
Aromatase over-expression in rodents
Recently a transgenic line of mice overexpressing aromatase enzyme (AROM+) has been developed (69, 70). These mice show highly elevated serum estradiol concentrations, with a reciprocal decrease in testosterone concentrations. The AROM+ males display several of the changes observed in males perinatally exposed to estrogens, such as undescended testes, testicular interstitial cell hyperplasia, hypoandrogenism, and growth inhibition of accessory sex glands. A disruption of spermatogenesis has also been observed which could be a consequence of multiple factors, including cryptorchidism, abnormal Leydig cell function, hypoandrogenemia or hyperestrogenemia. Estrogens are thought to inhibit Leydig cell development, growth and function, resulting in the suppression of androgen production (see 13 for review). The observation of numerous degenerating germ cells and the absence of spermatids within the seminiferous tubules of AROM+ mice suggest that germ cells development was arrested at the pachytene spermatocyte stage in the cryptorchid testes. Interestingly, the spermatogenic arrest occurred at a stage where P450arom is typically expressed. The spermatogenic arrest found in the AROM+ mice could be explained, at least partially, by the suppression of FSH action. The reduced serum FSH levels in AROM+ males are further evidence of the inhibiting actions of estrogens on FSH secretion in males. No significant differences in the LH concentrations were seen in AROM+ and wild type mice (69, 70).
Exposure to excess estrogens in humans
The clinical use of diethylstilbestrol (DES) by pregnant women in order to prevent miscarriage resulted in an increased incidence of genital malformations in their sons (71). In these individuals the presence of Müllerian ducts remnants was found indicating that fetal exposure to DES may have an effect on sex differentiation in men, as is the case in rodents (67). Moreover a large number of structural and functional abnormalities were found, the most frequent being: epididymal cysts, meatal stenosis, hypospadias, cryptorchidism and microphallus (71). The frequency of abnormalities was dependent on the timing of estrogen exposure: in fact, men who were exposed to DES before 11th week of gestation (i.e. the time of Müllerian ducts formation) had a two fold higher rate of abnormalities than those who were exposed only later (71). This data supports the previously discussed hypothesis that the asynchrony between formation and regression of embryonal reproductive structures is determined by estrogen exposure.
Various reports have demonstrated that semen quality of men exposed to DES in utero is significantly worse than in unexposed controls (72, 73). However, the sperm concentrations of most of the DES exposed men were well above the limit at which subfertility occurs, and it is therefore not surprising that the fertility of these men was reported to be normal (7). The risk of testicular cancer among men exposed to DES in utero has been a controversial issue and several meta-analyses showed no increased risk (74). However more direct evidence will be necessary in order to fully understand this issue.
While various studies suggest that environmental estrogens affect male fertility in animal models, the implications for human spermatogenesis are less clear (75). It has been demonstrated that male mice whose mothers have consumed a 29 ng/g dose of bisphenol A for seven days during pregnancy had a 20% lower sperm production as compared to control males (76). Various abnormalities in reproductive organs have also been described in males exposed to bisphenols (i.e. a significant decrease in the size of the epididymis and seminal vesicles and an increase in prostate gland volume), suggesting that bisphenols interfere with the normal development of the Wolffian ducts in a dose-related fashion. Exogenous estrogens could interfere with the development of the genital structures if administered during early organogenesis, by leading to both an impairment of gonadotropin secretion and by creating an imbalance in the androgen to estrogen ratio, which may account for impaired androgen receptor stimulation or inhibition according to the dose, the cell type and age (71, 77, 78, 79).
An excess of environmental estrogens has been suggested as a possible cause of impaired fertility in humans (50, 51). A progressive decline in sperm count has been reported in some Western countries during the past 50 years, suggesting a possible negative effect of environmental contaminants on male reproductive function (6, 51, 71, 77). Data concerning the role of estrogens in male reproductive structure development remains conflicting. Animal studies suggest that exposure to estrogen excess may negatively affect the development of reproductive male organs. These effects, however, are considered to be the result of an impaired hypothalamic-pituitary function as a consequence of estrogen excess and of the concomitant androgen deficiency (78, 79). Much of the knowledge on excess estrogen exposure and human fertility depends upon animal data and the validity of these concepts to humans has not been established.
Aromatase over-expression in humans
In 1996 a boy with aromatase excess syndrome was reported (80). His condition was presumably inherited in an autosomal dominant fashion with sex-limited expression as his father had a history of peripubertal gynecomastia, elevated serum estrogen levels and increased aromatase activity in vitro. The father was fertile and had a normal libido despite a small testicular volume (15 mL bilaterally), and a reduced testosterone level of 234 ng/dL(80). In the son, mild suppression of testicular growth and Leydig cell function probably reflected direct estrogen negative feedback on pituitary gonadotropin secretion. In general, the inhibitory effects of estrogen on reproductive function appear to be milder in males with aromatase excess syndrome than in patients receiving exogenous estrogens or with estrogen-secreting tumors, probably because serum estradiol and/or estrone levels are lower in the former (80).
Estrogen deficiency: human models
Effect of estrogen deficiency on gonadotropin feedback
The role of estrogens in human male physiology has become better understood as a result of the description of a man lacking a functional estrogen receptor a (4). This patient presented with tall stature, continuing linear growth during adulthood, unfused epiphyses and osteoporosis. He had normal serum testosterone, high estradiol and estrone levels, and increased FSH and LH concentrations. He had normal male genitalia with bilaterally descended testes each of 20-25 mL volume and a normal prostate volume. No further studies were performed on the reproductive system of this male (Table 5). The only data available are those on sperm characteristics, which revealed a reduced viability of sperm (Table 5).
Other important information about the role of estrogens in the human male came from the discovery of naturally occurring mutations in the aromatase gene. To date only five different cases of human male aromatase deficiency have been found, three of these males were discovered to be aromatase deficient during adulthood and one of them as a child (44-48, 63, 64).
The hormonal pattern of the four adult patients affected by aromatase deficiency is summarized in Table 5. The study of men with aromatase deficiency shows that estrogens modulate gonadotropin feedback by regulating both basal secretion of gonadotropins and their responsiveness to GnRH ( 44, 48, 63, 65, 66).
In the young patient with congenital aromatase deficiency no alterations were found either in gonadotropin secretion or in testis size, both testes were descended and the penis was normal (64). The presence of normal levels of gonadotropins raises the possibility that the role of estrogens in the regulation of the hypothalamo-pituitary-testicular axis becomes relevant in a later stage of life than infancy. This is in contrast to aromatase deficient female patients in whom an elevation of FSH and LH is seen even in childhood (85), demonstrating the importance of estrogens in the feedback regulation of gonadotropin secretion in girls during every stage of life.
Effect of estrogen deficiency on the human testis
Two of the adult males affected by aromatase deficiency showed a decreased testicular volume, one had normal testes, while the other had large testes. The histological study performed in only two of the four patients showed profound alterations in germ cell development, in particular one of the two patients had germ cells arrest at primary spermatocytes and the other had complete depletion of germ cells. Sperm analysis of one of these two patients showed severe oligozoospermia and astenozoospermia (Table 5). It remains unclear as to whether their disordered estrogen physiology accounts for the spermatogenic defects.
Effect of estrogen deficiency on the development of reproductive structures
Bilateral cryptorchidism was present in one patient with aromatase deficiency (45, 46), suggesting a possible role of estrogen also in testis descent, although this was not seen in the transgenic mice models (see above). The presence of a unique case of cryptorchidism among men with aromatase deficiency does not permit any conclusions to be drawn concerning a possible relationship between estrogen deficiency and the occurrence of abnormalities in testis development and descent.
ESTROGEN AND MALE SEXUAL BEHAVIOR
Gender-identity and sexual orientation
Sex steroids and particularly testosterone are able to affect adult male sexual behavior in mammals (86). In non-primate mammals, androgen exposure during late fetal and early neonatal development in the male accounts for the sexual dimorphism of the central nervous system (CNS), probably as a result of testosterone aromatization in the brain (87-92). Prenatal and perinatal estrogen action in the brain is believed to be responsible for the establishment of a male brain (93). Paradoxically, male rat brain is exposed to a greater amount of estradiol than female brain, since ovaries release less estrogen than testes at this stage of development (obviously in male estrogens derives from the conversion of testosterone produced by testis). Furthermore, estrogens are inactivated in the female fetus by various biochemical mechanisms, such as binding to alpha-fetoprotein (94). As a consequence, a sexual dimorphism of hypothalamic structures develops in rodents and the same mechanism seems to be involved also for the establishment of differences in hypothalamic structures between men and women (92, 95-97).
The role of sex steroids and of testosterone aromatization in the determination of the imprinting of sexual behavior has been considered of primary importance for the determination of both adult sexual orientation and sexual behavior in both animals and humans (92, 96-98). Particularly, it was thought that testosterone deficiency and the lack of its estrogenic metabolites during early phases of development could affect sexual orientation (92, 95, 96). Recently, a detailed study of a man with aromatase deficiency did not reveal any abnormalities of both gender-identity and sexual orientation (99). Based on this study the patient was categorized as masculine, his gender identity was male and the psychosexual orientation was heterosexual. Also data from the other men with aromatase deficiency don't show any association between congenital aromatase deficiency and gender-identity or sexual orientation disturbances (4, 45-48) (Table 7). Since aromatase deficient patients would be subjected to maternal estrogens in utero, it is also possible that such estrogen exposure would be sufficient for sexual behavior development.
These data suggest that congenital aromatase deficiency does not affect psychosexual orientation and gender-identity in humans and that, in contrast to animals, human psychological and social factors may be the most relevant determinants of gender role behavior in men, with hormones having a minor role. Hormones, in fact, may affect sexual differentiation and sex assignment at birth and, only indirectly, psychosexual development in men (79).
Sexual behavior
In mammals, adult male sexual behavior is at least partially dependent on the presence of testosterone. Androgens are also necessary for male sexual behavior during adult life (100-102). In fact, the lack of testosterone frequently produces loss of libido and erectile dysfunction (101, 102). At the same time, testosterone replacement therapy increases sexual interest and improves sexual behavior (86, 101). By contrast, the role of aromatization in the establishment and maintenance of male sexual behavior has been characterized only recently.
Congenital aromatase deficiency and estrogen action blockade result in a severe impairment of sexual behavior in rodents. ArKO mice (41) exhibit a significant reduction in mounting frequency and a significantly prolonged latency to mount when compared with heterozygous and wild-type animals (103). Also the sexual behavior of aERKO mice is characterized by a reduction of intromissions, an increase in the latency to first intromission and a lack of ejaculation, despite the presence of a normal motivation to mount females. The same sexual behavior pattern occurs in abERKO male mice (see 3, 13 for review). On the contrary, bERKO mice showed all three components of sexual behavior including ejaculation (Figure 7). These findings suggest that at least one of the ERs (ERa) is required for the expression of simple mounting behavior in male mice and, as a consequence, that activation of the androgen receptor alone is not sufficient for a fully normal sexual behavior, confirming that aromatization of androgens is also required.
Figure 7: Sexual behavior in estrogen deficient male mice.
The role of estrogens in male sexual behavior is confirmed by studies in gonadectomized rats treated with testosterone (104). Vagell and McGinnis showed, in fact, a complete inhibition of male sexual behavior in gonadectomized rats when the aromatase inhibitor fadrozole was administered in addition to testosterone, demonstrating that this inhibition disappeared when estrogen administration was added (104).
Much less is known about the role of estrogens in sexual behavior in the human male, particularly the degree to which the effects of testosterone ought really be ascribed to its conversion into estradiol. Previous studies aimed at addressing this issue have provided conflicting results both in support (105, 106) and against (107, 108) an important role of estrogen on human male sexuality. In order to evaluate the role of estrogens in human male sexual behavior, sexual activity has been investigated in a man with aromatase deficiency, before and during testosterone or transdermal estradiol treatment. When the patient received his physiological dose of estrogens (i.e. 25 mg transdermal estradiol twice weekly) he experienced an increase of all the parameters of sexual activity (the frequency of masturbation, sexual intercourse, erotic fantasies and libido) (Table 7, Figure 8), without change during testosterone treatment.
In men with congenital estrogen deficiency it seems that estrogen may play a role in adult sexual behavior, even if it's not possible to exclude that the improvements observed were the result of an improvement in well being and mood related to the estrogen replacement therapy.
Figure 8: Sexual behavior parameters in a men with aromatase deficiency:
effect of testosterone and estradiol treatment.
PHASE A: before testosterone treatment.
PHASE B: during testosterone treatment (testosterone enanthate): 250 mg every 10 days i.m for 6 months.
PHASE C: before estradiol treatment.
PHASE D: during transdermal estradiol treatment: 50 mg twice/week transdermal estradiol for 6 months.
PHASE E: during transdermal estradiol treatment: 25 mg twice/week transdermal estradiol for 9 months.
PHASE F: during transdermal estradiol treatment: 12,5 mg twice/week transdermal estradiol for 9 months.
These findings from transgenic mice and humans deficient in aromatase suggest that physiological levels of estrogens could be required for completely normal sexual behavior .
CONCLUSIONS
Sex steroids account for sexual dimorphism because they are responsible for the establishment of primary and secondary sexual characteristics, which are under the control of androgens and estrogens in male and female, respectively. (Figure 9).
Figure 9: Direct and indirect (estrogen-mediated) testosterone action.
Advances in the understanding of the role of estrogens in animal and human models suggest a role for this sex steroid in the reproductive function of both sexes. The fact that both estrogen excess and estrogen deficiency influence male sexual development, testis function, the hypothalamic-pituitary-testis axis, spermatogenesis and ultimately male fertility highlight the importance of estrogen action in the male. From an evolutionary perspective this provides an example of the parsimony operating in biological events which are crucial for the evolution of the human species such as growth and reproduction.
This chapter has been concerned with the reproductive effects of estrogens in males but there are emerging roles for estrogens in non-reproductive tissues. In particular, while traditionally, testosterone has been considered the sex hormone involved in bone maturation and growth arrest in men, but recently the key role of estrogens on growth has been emphasised (5, 44). In men and women, in fact, epiphyseal closure and growth arrest are not achieved without estrogens, underlining the fact that estrogens on human growth are highly conserved in both sexes. Thus it is clear that testosterone might act directly or through its conversion into estrogens (109). This aspect of estogen action is discussed in Chapter 2 and Chapter 3 in this section.
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