Low-density cDNA array-coupled to PCR differential display identifies new estrogen-responsive genes during the postnatal differentiation of the rat hypothalamus.
To identify estrogen (E)-responsive genes that may play important roles in the sexual differentiation and maturation of the neuroendocrine hypothalamus, we used mRNA differential display PCR to analyze hypothalamic RNA derived from estrogen-sterilized rats (ESRs). Neonatal rats were s.c.-injected with 100 microg of 17 beta-estradiol-benzoate (EB) for 5 days. Approximately 300 out of more than 2000 RNAs examined displayed a differential expression pattern between hypothalami of the ESR females compared to their 60-day-old controls. EB-dependent expression of these genes was further analyzed by low-density cDNA array using cDNA probe sets reverse-transcribed from the same groups; 98 genes were confirmed to be differentially expressed. We selected 41 clones that showed higher density differences between the two probe sets than mean density difference in control cyclophilin cDNA blots in the cDNA array. After being cloned into pGEM-T vectors, their sequences were analyzed. Homology searches identified four genes as a protein kinase C (PKC)-binding protein, NELL2 (clone 6-1), a thyroid nuclear factor, TTF-1 (9-1), Munc18-1 (17-6), and leuserpin-2 (18-5). The other 22 genes were similar to reported genes or cDNAs such as mouse kinesin-associated protein 3 (KAP3, 8b), mouse IgE binding lectin (15-1), normalized rat brain cDNA (5-1), rat cDNA (8-1) and rat embryonic cDNA (17-1). Fifteen clones such as clone 7-3 showed no match in the GenBank Database. Further characterization of eight clones (17-1, 7-3, 8-1, 5-1, NELL2, KAP3 homolog, IgE binding lectin homolog, and TTF-1) showed that their expression in the adult female rat hypothalamus is sensitive to neonatal treatment with EB. They showed brain-specific expression and moreover, showed an increase in their mRNA level before the initiation of puberty. Some of them showed gender differences in their different postnatal expression pattern. We speculate that further study will demonstrate that many of the E-regulated genes identified in the present study play important roles in the regulation of the sexual differentiation and E-dependent maturation of the hypothalamus.
Choi
E J
EJ
Department of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan 680-749, South Korea.
Ha
C M
CM
Choi
J
J
Kang
S S
SS
Choi
W S
WS
Park
S K
SK
Kim
K
K
Lee
B J
BJ
D000818:Animals; D000831:Animals, Newborn; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D007031:Hypothalamus / Q000254:growth & development* / Q000502:physiology; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D010053:Ovary / Q000254:growth & development; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012727:Sex Characteristics; D012733:Sex Differentiation; D014599:Uterus / Q000254:growth & development
D004967:Estrogens