Reducing Estrogen Synthesis Does Not Affect Gonadotropin Secretion in the Developing Boar

BIOLOGY OF REPRODUCTION 74, 58–66 (2006) Published online before print 14 September 2005. DOI 10.1095/biolreprod.105.043760 Reducing Estrogen Synthesis Does Not Affect Gonadotropin Secretion in the Developing Boar1 E.E. At-Taras,3 A.J. Conley,4 T. Berger,3 and J.F. Roser2,3 Department of Animal Science, 3 and Department of Population Health & Reproduction,4 School of Veterinary Medicine, University of California, Davis, California 95616 Boars have high concentrations of plasma and testicular estrogens, but how this hormone is involved in feedback regulation of the gonadotropins and local regulation of testicular hormone production is unclear. The present study examined the effects of reducing endogenous estrogens by aromatase inhibition on concentrations of plasma LH and FSH and on testicular and plasma concentrations of testosterone (T) and immunoreactive inhibin (INH). Thirty-six littermate pairs of boars were used. One boar from each pair was assigned to the control group (vehicle); the other boar to the treatment group (aromatase enzyme inhibitor, Letrozole, 0.1 mg/kg body weight [BW]). Weekly oral treatment started at 1 wk of age and continued until castration at 2, 3, 4, 5, 6, 7, or 8 mo. Plasma concentrations of gonadotropins, INH, T, estradiol (E2), and estrogen conjugates (ECs) were determined. Testicular tissue was collected at castration for determination of INH and T and for confirmation of reduced aromatase activity. The acute effects of aromatase inhibition on gonadotropins were monitored in two adult boars treated once with Letrozole (0.1 mg/kg BW). Treatment with the aromatase inhibitor reduced testicular aromatase activity by 90% and decreased E2 and ECs without changing acute, long-term, or postcastration LH and FSH. Plasma T, testicular T, and circulating INH concentrations did not change. Testicular INH was elevated in treated boars compared with controls. In conclusion, estrogen does not appear to play a regulatory role on gonadotropin secretion in the developing boar. This is in direct contrast to findings in males of several other species. estradiol, follicle-stimulating hormone, inhibin, luteinizing hormone, testosterone INTRODUCTION Estrogen is increasingly recognized as an important hormonal regulator of male reproductive function, as highlighted through studies with mouse knockout models. Estrogen-receptor knockout mice that are deficient in estrogen receptor alpha (ESR1) develop testicular atrophy with tubular degeneration and reduced sperm production and fertility [1, 2]. Researchers have demonstrated that this results from a lack of fluid reabsorption in the efferent ductules [3]. In contrast, MATERIALS AND METHODS 1 Supported by National Research Initiative Competitive grant 200235203-12606 from the USDA Cooperative State Research, Education, and Extension. 2 Correspondence: Janet F. Roser, Department of Animal Science, University of California, One Shields Ave., Davis, CA 95616. FAX: 530 752 0175; e-mail: Animals Thirty-six littermate pairs of boars were used in the present study. The boars were born and raised at the University of California, Davis, Swine Facility and were from established lines developed from Durocs, Hampshires, Yorkshires, and Pietrains provided by PIC USA (a division of Sygen International). Experiments were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [22] and approved by the Animal Use and Care Advisory Committee at the University of California, Davis. One animal from each pair was assigned to the control group (dosed orally with corn oil). The other animal was assigned to the treatment Received: 11 May 2005. First decision: 17 June 2005. Accepted: 9 September 2005. Ó 2006 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 58 estrogen receptor beta (ESR2) knockouts are phenotypically normal [4], although the immunolocalization of the ESR2 in Sertoli cells, Leydig cells, and developing germ cells of rat testes is consistent with a role of estrogen and ESR2 in testicular function. Aromatase (CYP19A1) is the enzyme solely responsible for the bioconversion of androgens to estrogens [5]. The CYP19A1 knockout mice, the phenotype of which is caused by targeted disruption of the CYP19A1 gene, are incapable of synthesizing estrogens. Initially, these mice are fertile, but testis weight and spermatogenesis are compromised with advancing age [6]. In CYP19A1 knockout mice, estradiol (E2) levels are undetectable, whereas levels of testosterone (T), LH, and FSH are elevated [7]. The role of estrogen in the regulation of gonadotropins has been investigated in several species [8–11] and is believed to influence the secretion of LH and/or FSH. In adult male mice [12, 13], monkeys [13], and men [8], E2 has a suppressant effect, whereas in stallions [10, 11], it has a stimulatory effect on LH and/or FSH secretion. Gonadotropins are essential for testicular growth and development and for the support of testicular factors necessary for the initiation and maintenance of spermatogenesis [14]. An understanding of the underlying factors controlling gonadotropin secretion therefore is important to understanding male reproductive development. The importance of estrogen in boars is especially intriguing, because these animals have higher levels of estrogens than are found in males of most species. In fact, plasma estrogen levels are well above those in sows during estrus [15]. Numerous studies have assessed and profiled plasma reproductive hormones in the developing boar [16–21] and although all have confirmed relatively high estrogen levels, none have attempted to explore the role of estrogen as a regulator of hormone dynamics. The present study elucidates the importance of estrogen in the regulation of gonadotropin secretion and in the maintenance and regulation of T and inhibin concentrations in the developing boar. To our knowledge, these are the first experiments in pigs that use a potent and specific aromatase enzyme inhibitor (Letrozole, CGS 20 267; Ciba-Geigy) to evaluate the chronic reduction of estrogen on the hypothalamic-pituitary axis of the boar. ABSTRACT AROMATASE AND REPRODUCTIVE HORMONES IN THE BOAR Testicular Homogenization Tissue was homogenized in 2 ml of 0.1 M phosphate buffer (pH 7.0) containing 0.01% thimerosal (Sigma) using a Barnant mixer-motor and pestle (General Electric) for approximately 2–3 min or until tissue was completely ground. The suspension was then spun at 13 000 3 g in a Sorvall RC-5B centrifuge (Du Pont) for 20 min, and supernatant was collected and frozen at 208C until assayed for E2, T, ECs, INH, and protein concentration. Protein Assay Homogenized testicular samples were diluted 1:100 in 0.1 M phosphate buffer containing 0.01% thimerosal before protein determination using the Bradford Protein Assay method (Bio-Rad Laboratories, Inc.). Protein values were used to (...truncated)