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

Molecular and Cellular Endocrinology 193 (2002) 7 /12 www.elsevier.com/locate/mce

Effect of estrogen deficiency in the male: the ArKO mouse model Y. Murata *, K.M. Robertson, M.E.E. Jones, E.R. Simpson Prince Henry’s Institute of Medical Research, 246 Clayton Road, Clayton, Vic. 3168, Australia

Abstract 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 Cyp 19 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. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aromatase; ArKO; Estrogens

1. Introduction The formation of estrogens from C19 steroids is catalysed by aromatase cytochrome P450, the product of the CYP19 gene (Means et al., 1989) In addition to ovarian granulosa cells (Jenkins et al., 1993), aromatase is expressed in several tissue sites (Simpson et al., 1997), including the Leydig cells, Sertoli cells (in immature animals) and germ cells of the testis (Carreau et al., 1999), mesenchymal cells of adipose tissue (Agarwal et al., 1997), osteoblasts and bone chondrocytes (Sasano et al., 1997) and numerous sites in the brain (Sasano et al., 1998). These extragonadal sites of estrogen biosynthesis possess several fundamental features that differ from those of the ovaries. Principally, the estrogen synthesised within these compartments is most probably biologically active only at the local tissue level in a paracrine or intracrine fashion (Labrie et al., 1997). Thus these sources of estrogens play an important, but

* Corresponding author. Tel.: /61-3-9594-3573; fax: /61-3-95946125 E-mail address: [email protected] (Y. Murata).

hitherto largely unrecognised, physiological and pathophysiological role.

2. Organisation of the aromatase gene Aromatase is the enzyme responsible for the ratelimiting step in the conversion of androgens to estrogens. In humans, aromatase is encoded by the CYP 19 gene that is located on chromosome 15q21.2 (Chen et al., 1988). The nine coding exons are found in the 30 kb 3?-region, whereas the 5?-flanking region contains a number of alternative untranslated exons I driven by multiple promoters. These promoters are regulated in a tissue-specific manner and regulate expression of aromatase in the gonads, adipose tissues, chondrocytes and osteoblasts in bone, brain, skin, fetal liver and placenta (Simpson et al., 2000) and are found within a 90 kb region upstream of the coding region. For example, the proximal promoter is found 1 kb upstream of exon II and it is activated in the gonads, whereas promoter I.4, located 69 kb upstream exon II, drives aromatase expression in adipose tissue and bone. Analysis of the human genome sequence indicated that the most distally located placental promoter I.1 was found approximately

0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 0 9 0 - 4

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89 kb upstream of the coding region (Sebastian and Bulun, 2001).

3. Models of estrogen insufficiency Aromatase is crucial for the biosynthesis of estrogen, converting androstenedione and testosterone to estrone and 17b-estradiol, respectively. Estrogens act through estrogen receptor a, ERa (Walter et al., 1985) and estrogen receptor b, ERb (Mosselman et al., 1996) and its variants (Leygue et al., 1999). The two receptors are not isoforms of each other, but are the products of distinct genes located on separate chromosomes. The expression of the two receptors appears to be tissuespecific. The ERa is the dominant species expressed in uterus, liver, adipose, skeletal muscle, pituitary and hypothalamus, whereas ERb is the major form in ovary and prostate (Couse et al., 1997), as well as other regions of the brain including the limbic system, cerebellum and cerebral cortex (Shughrue and Merchenthaler, 2000). The role of estrogens in both female and male physiology has been studied using several biological models. One approach consists of targeted deletions in the ERa (aERKO) (Lubahn et al., 1993) or ERb (bERKO) (Krege et al., 1998) or both abERKO (Dupont et al., 2000; Couse et al., 1999), where the receptor action was neutralised. Another, more direct approach is the aromatase deleted model (ArKO) (Fisher et al., 1998; Honda et al., 1998; Toda, 2000) where the source of estrogens was removed. These have all revealed novel insights on the multiple physiological roles of estrogens.

4. Aromatase gene mutation in the human So far, deficiency of the aromatase enzyme has been described in nine patients, of which, six were females (Harada et al., 1992; Ito et al., 1993; Morishima et al., 1995; Portrat-Doyen et al., 1996; Mullis et al., 1997; Ludwig et al., 1998). In most cases, the mother suffered from virilisation in the third trimester, she had elevated androgen and low estrogens levels, resulting in facial hair and acne. The newborns have androgen-induced female pseudohermaphroditism, low plasma estrogens and elevated androgen levels. During puberty, secondary sex characteristics failed to develop and hypergonadotropic, hypogonadism and polycystic ovaries were observed. They also developed tall stature, osteopenia and a delay in skeletal maturation. However, only three male aromatase deficient patients (Morishima et al., 1995; Carani et al., 1997; Deladoey et al., 1999) and one aER deficient patient (Smith et al., 1994) have been described. Except for one aromatase deficient male, who is still a child, all three

have tall stature due to failure of epiphyseal fusion, delayed bone age, eunuchoid skeletal proportions and severe osteoporosis. In the estrogen-resistant man, testosterone concentrations were normal, but estradiol, estrone and gonadotropins levels were elevated. Testicular size was normal with sperm count but reduced sperm viability. After estrogen treatment both aromatase deficient patients had improved bone mineral density and complete epiphyseal closure was achieved. However, the effect of aromatase deficiency on reproductive fertility was not clear. The patient described by (Morishima et al., 1995), had macroorchidism and no semen analysis was available. However, testosterone, androstenedione, FSH and LH levels were elevated which returned to normal levels following 3 years of estrogen therapy. Interestingly, testicular volume was reduced from 34 to 28 ml after estrogen treatment. The second male patient, described by (Carani et al., 1997), was infertile, however his serum levels of testosterone and LH were within normal range, while FSH was elevated. Nonetheless, the levels were reduced after 6 months of estradiol treatment. Testicular size was small and severely oligozoospermic; estrogen treatment not changing the volume of the testis or improving fertility. However, his brother who has a normal CYP 19 gene was also infertile with azoospermia, suggesting that the infertility in this patient could be independent of his aromatase deficiency.

5. Aromatase gene mutation in mice The aromatase deficient mouse model, ArKO, was created through disrupting the Cyp 19 gene by homologous recombination as described by (Fisher et al., 1998) Two other ArKO models have been independently created by (Honda et al., 1998) and (Toda, 2000). 5.1. Reproductive phenotype The male ArKO mice have elevated levels of testosterone and LH but normal levels of FSH. In young animals (12 /14 weeks), the males are fertile and their testis morphology appears to be normal (Fisher et al., 1998). However they showed an age dependent disruption of spermatogenesis, despite no decrease in LH or androgens. At 18 weeks of age, one out of five mice presented with disrupted spermatogenesis and grossly dysmorphic seminiferous tubules (Robertson et al., 1999). By 1 year of age, ArKO mice showed evidence of severe disruptions to spermatogenesis and a significant reduction in testis weight. Histological analysis showed degenerated round spermatids and multinucleated cells, suggesting that the spermatogenic disruption occurred in early spermiogenesis. In these tubules elongated spermatids were not seen, suggesting that

Y. Murata et al. / Molecular and Cellular Endocrinology 193 (2002) 7 /12

round spermatids did not complete elongation and spermiation. Stereological analysis indicated a decrease in round spermatid number, possibly resulting from an increase in apoptotic cell death, as visualised by TUNEL assays. However, six out of seven ArKO mice displaying spermiogenic arrest still contained some normal tubules, suggesting heterogeneity in the disruption. These animals also have hyperplasia and hypertropy of the Leydig cells, with the epididymides showing reduced or complete absence of sperm. Quantitative histomorphometry revealed a marked decrease in the volume of seminiferous epithelium in 1-year-old ArKO mice as compared with wild type mice. However, no change in seminiferous tubule luminal volume was observed in ArKO mice (Robertson et al., 1999) whereas in the aERKO mouse dilated lumens were obvious. This was due to an interruption of fluid resorption by the efferent ductules of the epidiymis, resulting in an infertile aERKO mouse (Lubahn et al., 1993). This is in contrast to the bERKO mice who reproductively develop normally, grossly and histologically indistinguishable as young adults from their littermates. The sexually mature male and female bERKO mice are fertile and exhibit normal sexual behaviour, but females have fewer and smaller litters when compared with wild type (Krege et al., 1998). Expression of aromatase in the testis has been reported to be present in a number of sites including the Sertoli cells (in immature rat) the Leydig cells in mature rat and mouse, and also in spermatocytes, round and elongated spermatids of the mouse (Nitta et al., 1993; Janulis et al., 1996b,a). Both ERa and ERb are also found in various cells type within the male reproductive tract. ERa is present in the Leydig cells and the efferent ductules of the rat (Hess et al., 1997; Kuiper et al., 1997) while ERb has been demonstrated in rat Sertoli cells, late spermatocytes, and early round spermatids (Enmark et al., 1997; Saunders et al., 1997; van Pelt et al., 1999) and in mouse Leydig cells and elongated spermatids (Rosenfeld et al., 1998). The results from the aromatase and ER knockout models show an important correlation between estrogen and its receptors to germ cell development. Although ArKO male mice were capable of breeding, their fertility was compromised. They produced reduced numbers of litters and not all ArKO mice were fertile. Variability within the same colony has been observed. Only 50% (five out of ten) ArKO males were able to reproduce, with generation of a reduced number of offspring when compared with wild type litter size (Robertson et al., 2001). In another ArKO model the authors reported that ArKO male mice fertility was reduced to 14% (Toda et al., 2001b) and in the third ArKO model, a total infertility in the knockout population was reported (Honda et al., 1998). Furthermore the ArKO male sexual behaviour was severely impaired. The latency to first mount was significantly increased

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when compared with the wild type littermates. When placed with the females, males immediately interacted with the female, appearing to sniff her genital area, similar to the wild type. However, this soon ceased and the male withdrew. No attempt to mount was initiated during the 20 min observed (Robertson et al., 2001), similar results were observed by (Toda et al., 2001b) and (Honda et al., 1998). ArKO mice also lost their aggressive behaviour, which could be reversed by E2 supplementation in neonates (Toda et al., 2001a). This is similar to the situation observed in abERKO mice, but differs from the aERKO which displays mounting behaviour but not ejaculation, whereas the bERKO mice have normal sexual behaviour. It is evident that loss of estrogenic stimulation has severely detrimental effects on male sexual behaviour (Ogawa et al., 1999). Presumably the estrogen in question is that produced as a consequence of local aromatase action in the brain, utilising circulating testosterone as substrate (Simpson et al., 2000; Simpson and Davis, 2000).

5.2. Adipose phenotype Estrogens are also involved in lipid metabolism as the ArKO mice developed an age dependent increase in intra-abdominal adiposity. The gonadal fat pads were significantly larger in females and males after 12 weeks of age and 17b-estradiol replacement for 3 /7 week-old female ArKO mice was able to restore their fat depots to masses comparable to those of wild type littermates. The accumulation of adipose tissue was associated with hyperplasia and hypertrophy of the adipocytes with simultaneous decrease in lean mass and display of hypercholesterolemia, hyperleptinemia and insulin resistance. Nevertheless, these mice were not hyperphagic but were less physically active (Jones et al., 2000). An increase in total body fat was also observed after sexual maturation age (16-week-old) in aERKO and abERKO, but not bERKO mice. The obesity was positively correlated with increased leptin serum levels, and insulin resistance was also observed in aERKO mice (Ohlsson et al., 2000). A similar phenotype was also reported in human aromatase deficient patients, as reviewed by (Grumbach and Auchus, 1999). The lipid deposit was not restricted to the adipose tissue, as ArKO mice showed visible hepatic steatosis (Jones et al., 2000; Nemoto et al., 2000). Enzymes of fatty acid b-oxidation (long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase and medium-chain acyl-CoA dehydrogenase), were down-regulated in ArKO mice and could be restored to normal levels with 17b-estradiol treatment (Nemoto et al., 2000).

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5.3. Bone phenotype

Acknowledgements

Aromatase is known to be an important regulator of bone cell function. As mentioned above, patients with aromatase or ERa deficiency suffered from severe osteoporosis with increased bone turnover. (Oz et al., 2000) observed a sexual dimorphic response in bone of the ArKO mice. Both male and female ArKO mice showed osteopenia in the lumbar spine. Histologically, animals of both sexes showed an osteoporotic phenotype characterised by significant decreases in trabecular bone volume and trabecular thickness. However, in males, aromatase deficiency contributed to a clear suppression of bone formation as shown by significant reductions in osteoblastic, osteoid, and mineralising sufaces. Osteoporosis in females however was more consistent with increased bone turnover. Furthermore, femur growth as measured by length, was 9% shorter in ArKO males compared with WT littermates (P B/ 0.001), while no significant differences in femur length between females were found. The sexual dimorphic response was not consistent with findings in another ArKO mice model, where increased bone resorption by osteoclast activity was observed in both males and females, and the degree of bone loss in 32-week-old ArKO mice was more severe in females than males (Miyaura et al., 2001). Although differences in both reports exist, it is clear that aromatase deficiency has profound impact in bone formation and maintenance. These findings are further supported by the observations from the ER knockout models where male ERa and ER double knockout, but not bERKO mice, displayed decreased longitudinal and radial skeletal growth with pronounced cortical osteopenia. This demonstrated that ERa, but not ERb is the ER mediating the effects of estrogen on skeletal growth in the male (Vidal et al., 2000). Furthermore, a sexually dimorphic response was found in bERKO mice, where female bERKO had increased bone mineral content (BMC) compared with wild type but no difference was found in male bERKO BMC (Windahl et al., 1999).

The Victorian Breast Cancer Research Consortium Inc., Melbourne, Australia, US Public Health Service Grant R37AG 08174 and National Health and Medical Research Council Grant 169010 supported this work.

6. Conclusion While the distribution and regulation of the aromatase gene have been extensively studied in human tissues and in vitro, the description of human estrogen deficient patients and studies of the estrogen deficient and estrogens receptor knock-out models, have demonstrated that estrogen plays an essential role in the development and maintenance of fertility, in both females and males, and also has a major role in the regulation of lipid metabolism and bone remodeling.

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