Molecular Human Reproduction, Vol. 6, No. 3, 219-225,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatozoa |
Expression of protamine-1 and -2 mRNA during human spermiogenesis
1 Institut für Veterinäranatomie, Frankfurter Strasse 98, D-35392 Giessen, 2 Institut für Pathologie, Langhansstrasse 10, D-35392 Giessen, and 3 Institut für Anatomie und Zellbiologie, Grosse Steinstrasse 52, D-06097 Halle, Germany
Abstract
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-2 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 PRM-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.
human testis/in-situ hybridization/laser-assisted cell-picking/protamines/spermiogenesis
Notes
4 To whom correspondence should be addressed
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