Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars

BIOLOGY OF REPRODUCTION 81, 388–395 (2009) Published online before print 29 April 2009. DOI 10.1095/biolreprod.109.076331 Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars1 C.J. Corbin,3 T. Berger,4 J.J. Ford,6 C.E. Roselli,7 W. Sienkiewicz,8 B.C. Trainor,5 J.F. Roser,4 J.D. Vidal,3 N. Harada,9 and A.J. Conley2,3 gaining better insight into roles of P450arom in reproductive function. ABSTRACT Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OHtestosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the ‘‘gonadal/hypothalamic’’ porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozoletreated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of aromatase, brain, brain sexual differentiation, hypothalamus, isoform, neuroendocrinology, porcine, regional expression, regulation, sexual dimorphism, steroid hormones, testis INTRODUCTION The functional differences in the hypothalamic-hypophyseal axis in male and female mammals was recognized more than 70 years ago [1] and has been an issue of considerable interest to reproductive biologists and behaviorists ever since. Many of the organizational events contributing to the development of the male behavioral and neuroendocrine phenotype are thought to be related to the development of sexually dimorphic nuclei [2] that have been found in the hypothalamus of many species [3, 4]. Fetal or neonatal exposure to testosterone [5] directs neural organization and activates male reproductive behaviors that emerge after puberty [6, 7]. This is due at least in part to effects of estradiol and not testosterone itself [8]. The conversion of androgens to estrogens by the enzyme aromatase cytochrome P450 (P450arom), which is expressed in the brain, has a central role in organizing neuroendocrine function [9] and a variety of reproductive and social behaviors [10]. For instance, P450arom expression in the preoptic area of the hypothalamus is higher in rams that prefer females to male sexual partners [11]. The sexually dimorphic expression of P450arom in the mammalian [12] and avian [13] brain provides additional support for the importance of the enzyme in these processes. Males express higher levels of P450arom in the brain than females [9], especially in functionally important regions of the hypothalamus and limbic system [14]. The aromatase hypothesis has focused attention on local conversion of testosterone to estradiol and activation of estrogen receptors as key events in sexual differentiation of the brain [12, 15–17]. However, recent investigations have reemphasized the role of testosterone or possibly other androgens that make additional important contributions more directly through androgen receptor activation [18]. Therefore, exactly how sex steroids interact to mold the development and function of the mammalian sexually dimorphic nuclei and associated neural substrates is still not entirely clear. The reproductive consequences resulting from changes in sexual differentiation of the hypothalamus also remain poorly understood, particularly the role of P450arom. 1 Supported in part by National Research Initiative Competitive Grant No. 2008-35203-19082 from the USDA Cooperative State Research, Education, and Extension Service. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture (USDA) neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. 2 Correspondence: A.J. Conley, Department of Population Health & Reproduction, University of California Davis, Davis, CA 95616. FAX: 530 752 4278; e-mail: Received: 20 January 2009. First decision: 23 February 2009. Accepted: 15 April 2009. Ó 2009 by the Society for the Study of Reproduction, Inc. eISSN: 1259-7268 http://www.biolreprod.org ISSN: 0006-3363 388 Downloaded from www.biolreprod.org.by guest on 09 June 2024 Downloaded from https://academic.oup.com/biolreprod/article/81/2/388/2557927 Departments of Population Health & Reproduction,3 Animal Science,4 and Psychology,5 University of California Davis, Davis, California USDA,6 ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, Nebraska Department of Physiology and Pharmacology,7 Oregon Health and Science University, Portland, Oregon Department of Functional Morphology,8 University of Warmia and Mazury, Olsztyn, Poland Fujita Health University,9 Toyoake, Aichi, Japan PORCINE HYPOTHALAMIC AROMATASE CYTOCHROME P450 MATERIALS AND METHODS Tissues The study utilized mature postpubertal male and female Meishan and commercial crossbred pigs. Boars were of two ages (mean 6 SEM age, 242 6 3 days [n ¼ 8] and 302 6 2 days [n ¼ 9]). Females were generally older (mean 6 SEM age, 431 6 30 days), but the youngest overlapped in age with the older group of boars (age range, 292–486 days). Hypothalami, pituitaries, and (in the case of boars) testes were collected within 5 min of slaughter and frozen immediately on dry ice. Tissue was dissected using the optic chiasm rostrally, the mammillary bodies caudally, and the lateral sulci to a depth of the third ventricle. In addition, four adult boars received an intracardiac perfusion with 4% paraformaldehyde in PBS (0.1 M [pH 7.4]) immediately following slaughter. Brains from these animals were subsequent (...truncated)