Wednesday, August 4, 2004
10:30 AM–12:30 PM
(757) OLIGONUCLEOTIDE MICROARRAY ANALYSIS OF GENE EXPRESSION IN ANDROGEN-TREATED NEONATAL MOUSE TESTIS.
Zhou, Qing1, Shima, James1, Nie, Rong1, Friel, Patrick1, Griswold, Michael1, 1 Center for Reproductive Biology, Pullman, WA
ABSTRACT- Androgens are required for normal spermatogenesis in mammalian males. These hormones directly regulate testicular somatic cells that in turn support germ cell differentiation. However, genes under androgen regulation in testis are not well understood. In this study, neonatal male mice (8dpp) were used as a model to study androgen action in testis as evidenced by alterations in gene expression. Since these mice do not have an established hypothalamus-pituitary-testis axis, their endogenous testosterone level is very low and changes in gene expression shortly after testosterone proprionate (TP) treatment are likely to be exogenous androgen effects. Additionally, there are no advanced germ cells in mice of this age and secondary response from germ cells, if any, should be limited. Mice were treated with 0.5 mg of testosterone proprionate (TP), dihydrotestosterone (DHT) or vehicle (oil) by subcutaneous injection, and testes were harvested 4, 8, and 16h after treatment. Global gene expression was monitored using the Affymetrix GeneChip Mouse Expression Set 430. Analysis of androgen-induced gene regulation was performed using Affymetrix Microarray Suite version 5.0 software and Silicon Genetics GeneSpring software. Real-time RT-PCR was performed to confirm the microarray results. Comparisons were made between androgen and vehicle treated testis as well as the possible differential effects of TP and DHT. Our methodology was verified by the presence of previously characterized TP-regulated genes including pem in Sertoli cells and CYP17 in Leydig cells. Based on a minimum fold change of 2 between the control and TP treated samples, we have identified 131, 80 and 118 genes being up-regulated 4, 8 and 16h after TP treatment respectively, and 42, 80 and 132 genes being down-regulated at the corresponding time points. No significant difference was found between TP and DHT on gene expression in testis. Some TP-regulated genes were dissected into testicular cell-type specific groups by in silico analysis and TP-regulation of these genes in specific cell types was confirmed using various primary cell cultures and cell lines. The data from this study may serve as a foundation for hypothesis-driven experiments and provide insights into gene networks and pathways under androgen control in testis. Supported by 5U 54HD42454-02.
KEY WORDS: testis, neonatal, androgen, microarray