Androgen induces p130 mRNA expression in the neonatal rat hypothalamus Keisuke Yonehara, Masatoshi Suzuki, Keitaro Yamanouchi, Masugi Nishihara * Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan Received 19 August 2002; received in revised form 18 September 2002; accepted 24 September 2002 Abstract Our previous research using cDNA microarray analysis demonstrated that female rats displayed a higher p130 mRNA level than males in the hypothalamus at postnatal day (PN) 5. In the present study, it was shown that at PN3 males had a significantly elevated mRNA level over females, whereas at PN7 females displayed a higher expression level using a real- time reverse transcription-polymerase chain reaction. In situ hybridization analysis indicated relatively strong p130 mRNA signals in the ventromedial nucleus and the arcuate nucleus in the neonatal hypothalamus. Subcutaneous injection of 5a-dihydrotestosterone as well as testosterone propionate to PN2 neonatal rats significantly increased p130 gene expression at PN3, whereas estradiol benzoate did not have a significant effect. These results suggest that expression of the p130 gene in the neonatal rat hypothalamus is responsive to androgens and may be involved in sexual differentiation of the brain. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: p130; Androgen; Gene expression; Sexual differentiation of the brain; Hypothalamus; Real-time reverse transcription-poly- merase chain reaction Sexual differentiation of the rodent brain is recognized to involve the transcriptional activation of multiple genes induced by gonadal steroids secreted during the perinatal period, known as the critical period [5]. In the male rat, testosterone secreted from testis induces the masculinization of the brain, whereas in the female, the relative absence of testosterone results in differentiation of the brain into the female type [2,5]. We have previously identified the granu- lin precursor gene as an androgen-inducible gene in the rat hypothalamus during the critical period [12] and demon- strated that this gene is involved in sexual differentiation of the rat brain [9–12]. We have further identified the p130 gene as one of the female-enriched genes in the hypothalamus at postnatal day (PN) 5 by cDNA microarray analysis [16]. p130 is a member of the retinoblastoma protein family (Rb, p107, p130), all of which are growth suppressors that interact with the viral oncoproteins and the E2F family proteins [17]. In the embryonic brain, p130 mRNA is expressed in proliferative neural progenitors and postmitotic neurons [17]. In neuroblastoma cells, p130 protein is upre- gulated at the late differentiation stages and its overexpres- sion induces differentiation [8]. These findings indicate that p130 is important for regulating neural differentiation. Sex steroids are known to influence many cellular events including neuronal differentiation and apoptosis [1], in which p130 has been implicated. We therefore suspected that the p130 mRNA expression level may be regulated by gonadal steroids in the neonatal rat hypothalamus. In the present study, we examined developmental p130 gene expression during the perinatal period and localization of the mRNA and protein in the hypothalamus by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry, respectively. Next, we determined the effects of testosterone and its metabolites, estradiol and dihydrotestosterone, on the p130 mRNA expression levels in the neonatal rat hypothalamus. Wistar–Imamichi rats were housed in a temperature- and light-controlled room (23 ^ 1 8C; lights on 05:00–19:00 h) with food and water ad libitum. Pregnant females were prepared and allowed to deliver normally. To determine the p130 gene expression pattern in the hypothalamus of intact male and female rats during the perinatal period, Neuroscience Letters 334 (2002) 107–110 0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(02)01114-X www.elsevier.com/locate/neulet * Corresponding author. Tel.: 181-3-5841-5386; fax: 181-3- 5841-8017 E-mail address: amnishi@mail.ecc.u-tokyo.ac.jp (M. Nishihara).