Identification of transcripts by macroarrays, RT-PCR and in situ hybridization in human ejaculate spermatozoa.

Round spermatids contain high levels of extremely varied mRNAs that are synthesized either throughout early spermatogenesis or during spermiogenesis from the haploid genome. Concomitantly, with major changes in the chromatin organization, arrest of transcription occurs at midspermiogenesis. However, previous investigations using RT-PCR have revealed the persistence of numerous and different transcripts in ejaculated spermatozoa. In the present study, a step-by-step analysis by means of macroarray hybridization, RT-PCR and in situ hybridization was performed to identify more accurately the different mRNA species found in the human ejaculated spermatozoa. The data showed an extended pattern of various transcripts encoding a diverse range of proteins involved in signal transduction and cell proliferation. For the first time, they demonstrated that mRNAs coding for the transcription factors NFkappaB, HOX2A, ICSBP, protein kinase JNK2, growth factor HBEGF and receptors RXRbeta and ErbB3 accumulate within the sperm nucleus. The origin and fate of the sperm transcripts remain subject to discussion.

Expression of the TAR RNA binding protein in human testis.

In testis, several RNA binding proteins have been shown to play a role in the translational regulation of specific transcripts. The human protein TRBP (TAR RNA binding protein) is the homologue of the mouse Prbp (Prm-1 RNA binding protein) involved in the protamine mRNA translational delay. TRBP is known to activate the HIV-1 long terminal repeat but this protein has never been investigated during spermatogenesis. The aim of this work was to analyse the TRBP expression in human testis. By Northern blot analysis, we demonstrated a major 1.5 kb transcript present at a high level in human testis and, to a lesser extent, in some other tissues. In-situ hybridization revealed that this transcript was present only in elongating spermatids. Antibodies raised against a 27 amino acid TRBP-specific peptide revealed a single protein of 43 kDa expressed in the cytoplasm of elongated spermatids. At the ultrastructural level, quantitative analysis of both TRBP mRNA and protein, using electron microscopy in-situ hybridization and immunocytochemistry, showed that TRBP is expressed mainly in spermatids at steps 3-4 of spermiogenesis. These results are in agreement with the probable role of TRBP in the control of human protamine mRNA translation.

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.

Electron microscopic in situ hybridization study of simultaneous expression of TNP1 and PRM1 genes in human spermatids.

Nuclear changes in the basic nucleoprotein complement occur during human spermiogenesis. Somatic type histones are displaced by transition proteins which are replaced themselves by protamines, the major nuclear proteins found in late spermatids and spermatozoa nuclei. Digoxigenin or Biotin labeled probes, coding respectively for human transition protein 1 (TP1) and protamine 1 (HP1), were used for double EM in situ hybridization. Immunodetection of hybrids with specific antibodies coupled to colloidal gold particles of different size (10 nm and 15 nm) was performed on the same preparations. Quantitative analysis of the nuclear and cytoplasmic labeling densities for the mRNAs coding for TP1 and HP1 showed the presence of transcripts in both the nucleus and cytoplasm of round spermatids until the elongation phase. Transcripts accumulated in the spermatid cytoplasm without any particular cellular compartmentalization. At the end of the spermatid elongation phase, the disappearing of TP1 and HP1 transcripts may be related to the arrest of transcriptional activity while the deposition of transition proteins and protamines successively occurs within spermatid nuclei.

Ultrastructural localization of rDNA and rRNA by in situ hybridization in the nucleolus of human spermatids.

The ultrastructural localization of rDNA and rRNA within the nucleolus of human spermatids was investigated by in situ hybridization at steps 1 and 2. Two different digoxigenin-labeled human probes from the rRNA transcription unit were used. Identification of hybrids was performed with immunogold techniques. Comparative observations in the Sertoli cell nucleolus as controls revealed that rDNA was predominantly visualized in the threads of the dense fibrillar component, while rRNA was detected over both the fibrillar component and the granular component. Within the nucleolus of round spermatids in the same sections of seminiferous tubules, rDNA labeling was localized over the spherical or stranded dense fibrillar components. rRNA labeling was found not only over these components but also in the adjacent nucleoplasm rich in ribonucleoprotein particles. These results are consistent with the view that the round spermatid nucleolus is transcriptionally active.

Cytochemical variations in the nucleolus during spermiogenesis in man and monkey.

The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate: EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15-20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of regional proteins in tissues and fluids in the human epididymis.

The proteins of epididymal tissues and fluids recovered from five regions of the human epididymis were separated by polyacrylamide gel electrophoresis under denaturing conditions. Among the 60 peptides identified, eight appeared to be expressed solely in the epididymal duct when compared to serum and testis proteins. Three of these (92, 47 and 24 Kd) showed a degree of regional specificity in fluids. The 92 Kd peptide was found in the caput and proximal corpus, the 47 Kd in the distal corpus and cauda and the 24 Kd in the caput of the epididymis. Three of the specific epididymal proteins (39, 30, 26 Kd) displayed a remarkable analogy to those found in man and monkey in other conditions and which are present at the sperm surface in the epididymis cauda.

[Ultrastructural labeling of the chromatoid body and the centriole-associated body using the DNase-gold colloidal complex in monkey spermatids]. / Marquage ultrastructural du corps chromatoïde et du corps associé au centriole à l'aide du complexe DNase-or colloïdal dans les spermatides de singe.

In Monkey spermatids at different steps of spermiogenesis, the use of DNase-gold complex showed, at the ultrastructural level, a labeling over the chromatin and concomitantly over the chromatoid body, centriole associated body and annular chromatoid body. The results obtained with the DNase-gold complex containing either DNA or actin led to discuss the nature of the substances revealed by the labeling in the cytoplasmic structures.

Autoradiographic investigation of sperm transit through the male mouse genital tract after tritiated thymidine incorporation.

The transit of spermatozoa in the genital tract of the male mouse was investigated by quantitative light microscopic autoradiography after intraperitoneal injection of tritiated thymidine. Transit duration in the caput and the corpus of the epididymis was shown to be 3 days; the total duration of transit in the genital tract was 5 days. These findings indicate that the time required for the transit of spermatozoa in the epididymal caput and corpus was comparable to that calculated in other mammals studied. However, the duration of sperm storage in the epididymal cauda appeared to be shorter than that previously reported for rodents.

High-resolution autoradiography of new formed proteins in the epididymis after incorporation of tritiated amino acids.

The synthesis and excretion of newly formed proteins in the principal cells of the head, body, and tail of the epididymis were studied by quantitative autoradiography in light and electron microscopy. Adult mice were killed from 5 min to 6 hr after intravenous injection of tritiated leucine, lysine, and arginine. The labels were taken up early and in greater amounts in the principal cells of the head. Radioactivity decreased in the cells of all three segments throughout the first hour following administration of the tracers. Thereafter, it increased in the lumen. High-resolution analysis showed successive peaks of relative concentration of the labels over the endoplasmic reticulum, Golgi apparatus, and apical plasma membrane, thus confirming that protein synthesis and excretion follow the usual pathway in the principal cells all along the epididymis. However, since a radioactivity peak occurred as early as 15 min over the apical membrane of cells in the head, it is likely that part of the endoplasmic reticulum-canalicular and poor in polysomes-is involved independently in the synthesis and rapid transport of newly formed proteins.

High resolution radioautographic study of newly formed protein in striated muscle with emphasis on red and white fibres.

The elaboration and distribution of newly formed proteins in the striated muscle of 21-day-old mice were investigated by quantitative radioautography at intervals between 2 and 240 min after intravenous injection of tritiated leucine. In radioautographs, the localization and the relative label concentration were comparatively estimated for the different components of mitochondria-rich fibres, in particular of red fibres, from the tibialis anterior muscle and of mitochondria-poor fibres from the oesophageal muscle. As early as 2 min after injection, radioactivity was detected over the nucleus, the polysome-rich sarcoplasm, the A and I bands, the Z lines, and the mitochondria in the two fibre types. Label localization did not change with time. The relative label concentration increased similarly in the polysome-rich sarcoplasm and the A and I bands of both fibre types within 30 min after injection, a confirmation that biosynthesis of myofibrillar proteins takes place rapidly. In each case, concentration was higher in the Z lines than in the I bands, and higher in the I bands than in the A bands, thus showing "in vivo" that the rates of synthesis of sarcomere protein components are not uniform. However, the relative label concentration was found to be higher in the Z lines of mitochondria-poor fibres than of mitochondria-rich fibres: this suggests that a higher synthetic rate of Z line protein, and probably of alpha actinin, is characteristic of the first type. Inversely, the concentration was higher in the mitochondria of mitochondria-rich fibres. This lead to the belief that high rate of protein synthesis in these organelles may account for the high rate of label incorporation into this type of fibre.