Margaret E. E. Jones

Estrogen and Spermatogenesis

Although it has been known for many years that estrogen administration has deleterious effects on male fertility, data from transgenic mice deficient in estrogen receptors or aromatase point to an essential physiological role for estrogen in male fertility. This review summarizes the current knowledge on the localization of estrogen receptors and aromatase in the testis in an effort to understand the likely sites of estrogen action. The review also discusses the many studies that have used models employing the administration of estrogenic substances to show that male fertility is responsive to estrogen, thus providing a mechanism by which inappropriate exposure to estrogenic substances may cause adverse effects on spermatogenesis and male fertility. The reproductive phenotypes of mice deficient in estrogen receptors alpha and/or beta and aromatase are also compared to evaluate the physiological role of estrogen in male fertility. The review focuses on the effects of estrogen administration or deprivation, primarily in rodents, on the hypothalamo-pituitary-testis axis, testicular function (including Leydig cell, Sertoli cell, and germ cell development and function), and in the development and function of the efferent ductules and epididymis. The requirement for estrogen in normal male sexual behavior is also reviewed, along with the somewhat limited data on the fertility of men who lack either the capacity to produce or respond to estrogen. This review highlights the ability of exogenous estrogen exposure to perturb spermatogenesis and male fertility, as well as the emerging physiological role of estrogens in male fertility, suggesting that, in this local context, estrogenic substances should also be considered male hormones.

The Role of Local Estrogen Biosynthesis in Males and Females

Natural (human) and experimental (mouse) models of estrogen insufficiency have revealed hitherto unexpected roles for estrogens in both males and females. In postmenopausal women, and in men, estrogen no longer has a major role as a circulating hormone, but rather it functions locally as a paracrine or even intracrine factor in tissue sites where it is formed. As a consequence, the tissue-specific nature of aromatase production assumes physiological and pathophysiological significance. The availability of circulating precursors is also important in sites where there is no local supply of C19 precursors, particularly in elderly women. The potential clinical significance of these findings in terms of the development of new therapeutic modalities is discussed.

Of mice and men: the evolving phenotype of aromatase deficiency

We are rapidly becoming aware of the importance of estrogen in maintaining virtually all facets of male health. In order for estrogens to be synthesized endogenously, the enzyme responsible for their synthesis from androgens, aromatase, must be functional. The seven known men in whom aromatase is nonfunctional all have a mutation in either exon V or IX of the CYP19 gene, which encodes aromatase. Collectively, these men are reported to have undetectable estrogen; normal to high levels of testosterone and gonadotropins; tall stature with delayed skeletal maturation and epiphyseal closure; osteoporosis; impaired lipid and insulin metabolism; and impaired reproductive function. The aromatase knockout mouse presents with a phenotype that is similar in many aspects and provides a valuable tool with which to examine and manipulate the actions of estrogen. By studying the naturally occurring aromatase-deficient humans, together with studies of the aromatase-knockout mouse, we are expanding our understanding of the essential role of estrogen in male physiology.

The ovarian phenotype of the aromatase knockout (ArKO) mouse

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21-23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.

Aromatase-deficient (ArKO) mice accumulate excess adipose tissue.

Aromatase is the enzyme which catalyses the conversion of C19 steroids into C18 estrogens. We have generated a mouse model wherein the Cyp19 gene, which encodes aromatase, has been disrupted, and hence, the aromatase knockout (ArKO) mouse cannot synthesise endogenous estrogens. We examined the consequences of estrogen deficiency on accumulation of adipose depots in male and female ArKO mice, observing that these animals progressively accrue significantly more intra-abdominal adipose tissue than their wildtype (WT) litter mates, reflected in increased adipocyte volume and number. This increased adiposity was not due to hyperphagia or reduced resting energy expenditure, but was associated with reduced spontaneous physical activity levels, reduced glucose oxidation, and a decrease in lean body mass. Elevated circulating levels of leptin and cholesterol were present in 1-year-old ArKO mice compared to WT controls, as were elevated insulin levels, although blood glucose was unchanged. Associated with these changes, the livers of ArKO animals were characterised by a striking accumulation of lipid droplets. Our findings demonstrate an important role for estrogen in the maintenance of lipid homeostasis in both males and females.

Recognizing rare disorders: aromatase deficiency

Aromatase deficiency is rare in humans. Affected individuals cannot synthesize endogenous estrogens. Aromatase is the enzyme that catalyzes conversion of androgens into estrogens, and if aromatase is nonfunctional because of an inactivating mutation, estrogen synthesis cannot occur. If the fetus lacks aromatase activity, dehydroepiandrosterone sulfate produced by the fetal adrenal glands cannot be converted to estrogen by the placenta, so is converted to testosterone peripherally and results in virilization of both fetus and mother. Virilization manifests as pseudohermaphroditism in female infants, with hirsutism and acne in the mother; the maternal indicators resolve following delivery. To date, only seven males and seven females with aromatase deficiency have been reported. Affected females are typically diagnosed at birth because of the pseudohermaphroditism. Cystic ovaries and delayed bone maturation can occur during childhood and adolescence in these girls, who present at puberty with primary amenorrhea, failure of breast development, virilization, and hypergonadotrophic hypogonadism. Affected males, on the other hand, do not present with obvious defects at birth, so are diagnosed much later in life, presenting with clinical symptoms, which include tall stature, delayed skeletal maturation, delayed epiphyseal closure, bone pain, eunuchoid body proportions and excess adiposity. Estrogen replacement therapy reverses the symptoms in male and female patients.

Effect of estrogen deficiency in the male: the ArKO mouse model

Aromatase, the enzyme responsible for the conversion of androgens to estrogens, is present in the mouse gonads, brain, adipose tissue and bone. Depletion of endogenous estrogens in the aromatase deficient mouse (ArKO) caused by the targeted disruption of the Cyp19 gene resulted in an impairment of sexual behaviour and an age-dependent disruption of spermatogenesis. This disruption occurred during early spermiogenesis, due possibly to increased number of apoptotic round spermatids. Development of obesity was associated with ageing, decrease in lean mass, hypercholesterolemia, hyperleptinemia, and insulin resistance and hepatic steatosis. However, it was not correlated with hyperphagia but to decreased physically-active behaviour. ArKO mice also developed osteoporosis. Thus, studies using the ArKO mice model has led to several insights into the multiple roles played by estrogens in the development and maintenance of fertility, sexual behaviour, lipid metabolism and bone remodelling.

Estrogen deficient male mice develop compulsive behavior

BACKGROUNDnAromatase converts androgen to estrogen. Thus, the aromatase knockout (ArKO) mouse is estrogen deficient. We investigated the compulsive behaviors of these animals and the protein levels of catechol-O-methyltransferase (COMT) in frontal cortex, hypothalamus and liver.nnnMETHODSnGrooming was analyzed during the 20-min period immediately following a water-mist spray. Running wheel activity over two consecutive nights and barbering were analyzed. COMT protein levels were measured by Western analysis.nnnRESULTSnSix-month old male but not female ArKO mice develop compulsive behaviors such as excessive barbering, grooming and wheel-running. Excessive activities were reversed by 3 weeks of 17beta-estradiol replacement. Interestingly, the presentation of compulsive behaviors is accompanied by concomitant decreases (p < .05) in hypothalamic COMT protein levels in male ArKO mice. These values returned to normal upon 17beta-estradiol treatment. In contrast, hepatic and frontal cortex COMT levels were not affected by the estrogen status, indicating region- and tissue-specific regulation of COMT levels by estrogen. No differences in COMT levels were detectable between female animals of both genotypes.nnnCONCLUSIONSnThis study describes the novel observation of a possible link between estrogen, COMT and development of compulsive behaviors in male animals which may have therapeutic implications in obsessive compulsive disorder (OCD) patients.

Estrogen regulates development of the somatic cell phenotype in the eutherian ovary

Steroids play a critical role in gonadal differentiation in birds, reptiles, and amphibia whereas gonadal differentiation in mammals is thought to be determined by genetic mechanisms. The gonads of female mice incapable of synthesizing estrogens due to disruption of the aromatase gene (ArKO) provide a unique model to test the role of estrogen in regulating the gonadal phenotype. We have shown that in the absence of estrogen, genetically female mice develop testicular tissue within their ovaries. The ovaries develop cells that possess structural and functional characteristics of testicular interstitial cells and of seminiferous tubule‐like structures lined with Sertoli cells. Moreover, the ovaries express mRNA for the testis‐specific Sertoli cell transcription factor Sox 9 and espin protein, which is specific for inter‐Sertoli cell junctions. The development of the testicular tissue in this model can be reverted/postponed by replacing estrogen. When ArKO female mice were fed a diet containing phytoestrogens, the appearance of Leydig and Sertoli cells was postponed and reduced. Furthermore, administration of estradiol‐17J decreased the number of Sertoli and Leydig cells in the ovaries. These findings constitute definitive evidence that estrogen plays a critical role in maintaining female somatic interstitial and granulosa cells in the eutherian ovary.—Britt, K. L., Kerr, J., ODonnell, L., Jones, M. E. E., Drummond, A. E., Davis, S. R., Simpson, E. R., Findlay, J. K. Estrogen regulates development of the somatic cell phenotype in the eutherian ovary. FASEB J. 16, 1389–1397 (2002)

Ovarian steroid receptors and their role in ovarian function

The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The precise role of oestrogen in the ovary remains to be elucidated, hence the data presented here which arises from studies designed to resolve this issue. Oestrogens signal via two receptor subtypes ERalpha and ERbeta, both of which are present in the ovary. ERbeta, the most abundant mRNA, is primarily expressed by GC where it transduces signals from ovarian-derived and exogenous oestrogens. Specific roles for each of the ERs in the ovary have yet to be established, despite ER knockout studies indicating both are required for normal function. The ArKO mouse is a model of oestrogen insufficiency. These mice are infertile as a result of arrested folliculogenesis (at the antral stage) and a failure to ovulate. Trans/re-differentiation of somatic cells in the ovary gives rise to Sertoli cell-like and Leydig cell-like cells within abnormal follicular structures. Disruption to the balance of sex steroids in the ovary is likely to facilitate this phenotype. Future studies will focus on the regulation of somatic cell differentiation, assigning roles to individual ERs and establishing definitive targets of oestrogen action in the ovary.