期刊
MOLECULAR HUMAN REPRODUCTION
卷 6, 期 3, 页码 219-225出版社
OXFORD UNIV PRESS
DOI: 10.1093/molehr/6.3.219
关键词
human testis; in-situ hybridization; laser-assisted cell-picking; protamines; spermiogenesis
During spermiogenesis, the histone-to-protamine replacement causes the compaction of the spermatid chromatin. The genes for protamines, PRM-1 and PRM-2, are transcribed in round and elongating spermatids, The transcripts are stored in a translationally-repressed state by the binding of protein repressors before being translated in elongating and elongated spermatids. RNA extracts from homogenized whole testis samples supply only average data, and cell-specific and stage-specific expression cannot be addressed. Therefore, we used UV-laser-assisted cell-picking (UV-LACP) to select spermatids of defined differentiation steps, Subsequent reverse transcription-polymerase chain reaction (RT-PCR) with intron-spanning primer pairs allowed the detection of DNA-free and pseudogene-free PRM-1 and PRM-P cDNA. Additional in-situ hybridization with digoxygenin-labelled cRNA probes exhibited PRM-1 and PRM-2 mRNA from step 1/2 spermatids to step 4 spermatids, but not in elongated spermatids. RT-PCR revealed amplicons for PRIM-1 and PRM-2 in all spermatids except step 3 round spermatids, Applying proteinase K digestion, PRM-1 and PRM-2 transcripts were also detected in step 3 spermatids indicating that protein repressors may bind to both PRM-1 and PRM-2 mRNA in step 3 round spermatids. These data demonstrate that the combination of UV-LACP and non-radioactive in-situ hybridization appear to be a suitable approach for the study of cell-specific and stage-specific gene expression during spermiogenesis.
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