Prenatal Hyperandrogenization Induces Metabolic and Endocrine Alterations Which Depend on the Levels of Testosterone Exposure Sabrina Amalfi 1 , Leandro Martı´n Velez 1 , Marı ´a Florencia Heber 1 , Susana Vighi 2 , Silvana Rocı´o Ferreira 1 , Adriana Vega Orozco 3 , Omar Pignataro 4 , Alicia Beatriz Motta 1 * 1 Laboratorio de Fisio-patologı ´a Ova ´rica, Centro de Estudios Farmacolo ´ gicos y Bota ´nicos, Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas, Universidad de Buenos Aires, Buenos Aires, Argentina, 2 Departamento de Patologı ´a, Hospital de Clı ´nicas, Buenos Aires, Argentina, 3 Laboratorio de Investigaciones Biome ´ dicas, Instituto de Biologı ´a y Medicina de Cuyo, Buenos Aires, Argentina, 4 Laboratorio de Endocrinologı ´a Molecular y Transduccio ´n de Sen ˜ ales, Instituto de Biologı ´a y Medicina Experimental, Nacional de Investigaciones Cientı ´ficas y Te ´ cnicas, Buenos Aries, Argentina Abstract Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogeniza- tion induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life. Citation: Amalfi S, Velez LM, Heber MF, Vighi S, Ferreira SR, et al. (2012) Prenatal Hyperandrogenization Induces Metabolic and Endocrine Alterations Which Depend on the Levels of Testosterone Exposure. PLoS ONE 7(5): e37658. doi:10.1371/journal.pone.0037658 Editor: Josep V. Planas, Universitat de Barcelona, Spain Received January 12, 2012; Accepted April 22, 2012; Published May 24, 2012 Copyright: ß 2012 Amalfi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by FONCyT-Argentina (BID 949/2006 and BID 71/2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: aliciabmotta@yahoo.com.ar Introduction Polycystic ovary syndrome (PCOS), one of the most common reproductive disorders, affects between 8 to 12% women in their reproductive ages [1]. Women with PCOS display oligo or anovulation, hyperandrogenism and/or ovarian cysts [2]. PCOS is frequently associated with hyperinsulinaemia, insulin resistance syndrome, increased cardiovascular risk and type 2 diabetes mellitus [3–5]. Its etiology remains uncertain, but current theories emphasize genetic and intrauterine origins coupled with environ- mental factors such as diet and altered lifestyle patterns [3]. A battery of animal models used for the study of PCOS has allowed focusing on different aspects of the pathology. In that context, prenatal androgen exposure is able to induce PCOS and metabolic syndrome in adult female rats [6–8], monkeys [9] and sheep [10–12]. In the brain, a prenatal excess of testosterone induces sex differences [9] and defeminization by increasing pulses of gonadotropin-releasing hormone (GnRH) [13]. In addition, a prenatal excess of testosterone increases body weight, induces insulin resistance [8] and deficiency of 21-hydroxylase [14] during the adult life. Recently, Legro et al [15] reported a direct association between birth weight and metabolic phenotypes in women with PCOS. However, data concerning the mechanisms involved in the prenatal excess of androgen and change in the secretion of hormones is still controversial [8,13,16]. Although it is well accepted that prenatally androgenized animal models help in investigating the etiology of PCOS, little is known about whether the androgen concentration of androgen determines the phenotype of PCOS during the adult life. Then, the aim of the present study was to determine whether different doses of testosterone at fetal life have different life-long effects. Prostaglandins (PGs) modulate different ovarian functions, such as the rupture of ovarian follicles associated with ovulation [17,18] and luteolysis [19,20]. In addition, PGE is increased in patients with PCOS [21]. We have previously reported that hyperandro- PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37658