title: Down-regulation of GAT-1 mRNA expression in the microdissected hypothalamic medial preoptic area of rat offspring exposed maternally to ethinylestradiol.
abstract: Steroid hormones are powerful regulators of gene transcription in the brain and have the potential to permanently alter the structure and function of the developing brain. Steroid-mediated altered gene expression may thus be responsible for the molecular cascade for sexual differentiation. In this study, to assess effects of maternal exposure to ethinylestradiol (EE) on brain sexual differentiation of offspring, region-specific mRNA expression of two estrogen-responsive genes, gamma-aminobutyric acid transporter type 1 (GAT-1) and anti-apoptotic bcl-xL was measured in the medial preoptic area (MPOA), including sexually dimorphic nucleus (SDN), at the late stage of brain sexual differentiation in rats. Pregnant Sprague-Dawley animals were fed diets containing EE at concentrations of 0, 0.02, 0.1, and 0.5 ppm from day 15 of pregnancy to day 9 after delivery. In another group, neonates were directly injected with estradiol benzoate (EB: 10 microg/pup, sc) on postnatal day (PND) 2. The MPOA on PND 9 was microdissected from methacarn-fixed paraffin-embedded brain sections to measure mRNA levels by competitive RT-PCR, followed by plate hybridization. EE-exposure decreased GAT-1 expression dose-dependently from 0.02 ppm in females and at 0.5 ppm in males, while EB-treatment caused reduction only in females. EE-exposure did not alter Bcl-xL levels. At week 11, EE-exposed females exhibited a similar spectrum of histopathological changes in endocrine-linked organs as with EB, evident from 0.1 ppm, while in males EE-exposure did not cause histopathological alteration despite clear change with EB-treatment. Measurement of SDN-POA dimensions at week 11 revealed volume reduction in males exposed to 0.5 ppm EE or EB. The results suggest that GAT-1 expression in the developing MPOA is a sensitive measure for the level of disruption of brain sexual differentiation due to maternal dietary exposure to estrogens, despite definite reproductive abnormalities may not be detectable in males with this exposure protocol.
authors.affiliation: Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. shibutan@nihs.go.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Shibutani
authors.forename: Naoya
authors.initials: N
authors.lastname: Masutomi
authors.forename: Chikako
authors.initials: C
authors.lastname: Uneyama
authors.forename: Naoko
authors.initials: N
authors.lastname: Abe
authors.forename: Hironori
authors.initials: H
authors.lastname: Takagi
authors.forename: Kyoung-Youl
authors.initials: KY
authors.lastname: Lee
authors.forename: Masao
authors.initials: M
authors.lastname: Hirose
mesh_terms: D000818:Animals; D001835:Body Weight / Q000187:drug effects; D015536:Down-Regulation; D004967:Estrogens / Q000633:toxicity*; D004997:Ethinyl Estradiol / Q000633:toxicity*; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D018507:Gene Expression Regulation, Developmental; D066298:In Vitro Techniques; D008297:Male; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D009929:Organ Size / Q000187:drug effects; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects; D011301:Preoptic Area / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012737:Sex Factors
chemical_list: D004967:Estrogens; D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C493415:Slc6a1 protein, rat; D004997:Ethinyl Estradiol