Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

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Abstract

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.

Introduction

Androgens are necessary for feedback regulation of gonadotropin secretion and for maintaining libido in males. For a long time, a connection between androgen and estrogen was not made because they produced their effects through two receptor systems and some of their actions were antagonistic. Later it was found that androgens also act as a prohormone for the local production of estrogens in target tissues. The aromatase enzyme complex (CYP19) has the unique ability to convert androgens to estrogen through a multistep enzymatic pathway [1]. Aromatase is present in a variety of tissues, including skin, adipose, bone, muscle, and brain, so that the relatively low serum levels of estrogens in males most likely do not accurately reflect their concentration in target cells and their importance for mediating local effects. Clinical experiments [2], [3], [4], [5], as well as endocrine evaluations of men that have mutations in the genes for aromatase or estrogen receptor [6], demonstrate that estrogen deficiency, either induced or naturally occurring, results in chronic elevations of gonadotropins in serum. This observation, which is indicative of a disrupted negative feedback mechanism, leaves little doubt that estrogens, and by inference aromatase, are crucial for the feedback control of gonadotropin secretion in men. However, gonadotropins are secreted in an episodic manner that is determined by the integrated response of both the hypothalamus and anterior pituitary to circulating steroids as well as environmental cues. For ethical and experimental reasons, it is not possible to access the hypothalamus, pituitary, or portal circulation in humans by directly measuring GnRH secretion or discretely inhibiting aromatase activity. Although a recent carefully designed and executed clinical study [7] implicates both the hypothalamus and pituitary as sites of estrogen’s action in men, it is impossible to directly and unequivocally prove that central aromatization is part of this process in humans. For this reason, an experimental animal model whose reproductive system is closely related to Homo sapiens is needed to address questions regarding the local production of estrogens from androgen precursors in the feedback regulation of gonadotropins. Experiments on laboratory rats suggest that aromatization of androgens to estrogens is not required for the suppression of gonadotropin secretion in rodents; although in rats this pathway is required for the expression of reproductive behaviors [8], [9]. In contrast, our research, and that of others, clearly establishes that negative feedback regulation of gonadotropins in non-human primates has both an androgenic and an estrogenic component [10], [11], [12], [13] and that the regulation of neural aromatase activity (AA) is of prime importance to this physiological process. The non-human primate has long been recognized as an important model for endocrinological investigations relevant to the human reproduction especially for studies of female reproduction, because of the similarities between the control of the ovarian cycle in women and female monkeys [14]. The present review summarizes studies on macaques performed in our laboratory that have helped clarify the role of aromatase and estrogens in the neuroendocrine regulation of male reproduction.

Section snippets

Evidence that estrogen and aromatase play important roles in negative feedback regulation of LH secretion in adult male rhesus monkeys

In non-human primates, testosterone and LH are secreted on a diurnal schedule [15]. Greater quantities of these hormones are secreted in the evening than in the morning hours [16]. After castration, gonadotropin concentrations gradually increase in the systemic circulation of rhesus macaques, but can be suppressed if large amounts of exogenous testosterone are administered [10], [17]. The earliest experimental indication that estrogens contribute to negative feedback regulation of gonadotropins

Sites of steroid action on LH

In the monkey, testosterone acts primarily, although probably not exclusively, on the brain to regulate LH secretion by suppressing the frequency of the GnRH pulse generator. This was first inferred from analysis of the frequency and amplitude of LH secretion in the peripheral circulation, but has not yet been confirmed by directly measuring GnRH secretion in males [20]. Thus, castration leads to an increased LH pulse frequency and amplitude, whereas testosterone replacement reduces LH pulse

Sites of estrogen synthesis

A substantial amount of estrogen is produced by both peripheral and central aromatization of androgens in vivo [28], [29], [30] making it difficult to determine the relative importance of these estrogen sources for the negative feedback regulation of LH secretion in male monkeys. However, unequivocal evidence that central aromatization is involved in the control of pulsatile LH secretion has recently been obtained in male sheep [31]. Infusion of the aromatase inhibitor fadrozole into the third

Distribution and regulation of aromatase mRNA in the monkey brain

To further investigate neural aromatase, we examined the expression of aromatase mRNA within the rhesus monkey brain [38], [39]. For our initial studies, we cloned a 455-nucleotide (nt) cDNA that spanned exons 3–5 of the coding region. Using this probe, we developed a ribonuclease protection assay and studied the distribution of aromatase mRNA in monkey brain. Two RNA fragments were protected by our assay, one was the expected full-length of 455-nt, and the other was truncated at 300-nt. Based

Testosterone insensitivity and aromatase activity

Male rhesus monkeys that have been castrated for more than 3 weeks are unresponsive to the negative feedback actions of testosterone [10], [17], [49]. Instituting a replacement regimen that produces circulating levels of testosterone that are within the physiologic range fails to suppress the hypersecretion of LH and FSH, unless estradiol is given concomitantly [10], [17]. This apparent resistance to the inhibitory action of androgen on gonadotropin secretion in the chronic agonadal state has

Conclusions

The central nervous system of the male monkey contains a heterogeneous distribution of neurons that can biosynthesize estrogen from androgen precursors supplied by the arterial system of the brain. Not only is androgen used as raw material for estrogen synthesis by these neurons but, it also exerts stimulatory effects on the aromatase mRNA, which serves as a template for production of the aromatase protein. Estrogens produced locally within the brain appear to be necessary components of

Acknowledgements

This work was supported, in part, by NIH grants HD18196 (JAR) and D43 TW HD00669 and NSF grant IBN 9817037 (CER).

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