Expression of protamine-1 and -2 mRNA during human 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-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.
Steger
K
K
Institut für Veterinäranatomie, Frankfurter Strasse 98, D-35392 Giessen, Germany.
Pauls
K
K
Klonisch
T
T
Franke
F E
FE
Bergmann
M
M
D000328:Adult; D000368:Aged; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008297:Male; D008875:Middle Aged; D011479:Protamines / Q000235:genetics*; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013087:Spermatids / Q000378:metabolism* / Q000473:pathology; D013091:Spermatogenesis
C082073:PRM1 protein, human; D011479:Protamines; D012333:RNA, Messenger; C068621:protamine 2