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RNA PROFILES IN RAT AND MOUSE EPIDIDYMAL SPERMATOZOA
Citation:
Thompson, K E., H Ren, J E. Schmid, AND D J. Dix. RNA PROFILES IN RAT AND MOUSE EPIDIDYMAL SPERMATOZOA. Presented at Society for the Study of Reproduction, Cincinnati, OH, July 19-22, 2003.
Description:
RNA PROFILES IN RAT AND MOUSE EPIDIDYMAL SPERMATOZOA
Kary E. Thompson1, Hongzu Ren1, Judith E. Schmid1 and David J. Dix1
1Reproductive Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, RTP, NC.
Mature spermatozoa are transcriptionally inactive, but can contain residual mRNAs expressed earlier in germ cell development. This study was designed to investigate the RNA profiles of rodent sperm by microarray and RT-PCR. Sperm were collected from the cauda epididimis of Sprague-Dawley rats (d75; n=3) or CD-1 mice (d65; n=3). Sperm were washed with a hypotonic solution (SDS/Triton X) to lyse somatic cells. Sperm RNA was isolated and then either amplified by in vitro transcription and labeled with Cy5 for microarray hybridization, or reverse-transcribed for PCR amplification. Rat and mouse RNA isolated from caudal sperm were demonstrated to be essentially free of 18S and 28S rRNA and enriched for mRNA by both agarose gel electrophoresis and Bioanalyzer LabChip (Agilent Technologies, Palo Alto, CA). On the mouse spotted-oligonucleotide microarray, hybridization signal (fluorescence) for 429 of 16,461 genes and ESTs (expressed sequence tags) was measured to be four times greater than background. In the rat array, 260 of 3898 genes and ESTs were found to be four times greater than background. With both mouse and rat arrays, numerous mRNAs that encode for proteins involved with sperm function (Dj1/SP22), meiosis (synaptonemal complex protein 1), cell cycle (cyclin B1), and spermatogenesis (transition protein 1, protamine 2, Dnajb3) were detected. On the more extensive mouse array, sperm mRNAs were also found for DAZ associated protein 1, heat shock 70kD protein 1-like, Hsp70-2, phosphoglycerate kinase 2, protamine 3, and transition protein 2. RT-PCR of mouse and rat sperm RNA also demonstrated amplicons for b-actin, Hsp70-2/Hspt70, protamine 2, and SP22/ Dj1. Thus rodent sperm contains mRNAs reflective of spermatogenesis and male fertility, suggesting the utility of sperm RNAs as biomarkers. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.)
KEY WORDS: Microarray, spermatozoa, RNA, rat, mouse