Co-localization of HP1 and TP1 transcripts in human spermatids by double electron microscopy in situ hybridization

Nuclear changes in the basic nucleoprotein complement occur during spermiogenesis in man. Somatic type histones are displaced by transition proteins which are replaced themselves by protamines, the major nuclear proteins present in late spermatids and sperm nuclei. Sense and antisense 35S-labelled riboprobes, coding respectively for human transition protein 1 (TP1) and protamine 1 (HP1), were synthesized with modified specific oligonucleotides and were used for light microscopy in situ hybridization. A double EM in situ hybridization was performed using a digoxigenin-labelled probe for TP1 and a biotin-labelled probe for HP1, and hybrids were revealed, respectively, with specific antibodies coupled to colloidal gold particles of different sizes (10 nm and 15 nm). For both types of transcripts, histological study revealed a specific distribution of the silver grains in the adluminal region of the seminiferous tubules where spermatids are localized. Quantitative ultrastructural analysis of the nuclear and cytoplasmic labelling densities for the mRNAs coding for TP1 and HP1 showed that the transcripts were found in both the nucleus and cytoplasm of round spermatids and persisted until the elongation phase. Transcripts accumulated in the spermatid cytoplasm without any particular cellular compartmentalization. At the end of the spermatid elongation phase, the disappearance of TP1 and HP1 transcripts may be related to the arrest of transcriptional activity, while the deposition of transition proteins and protamines occurs successively within spermatid nuclei.