The most important event determining the nuclear status of sperm cells is the replacement of histones by protamines, which are the basic nuclear proteins of mature spermatozoa. A first step in this exchange is the displacement of histones by transition proteins (TP). Our study demonstrates, for the first time, the sequential expression of the testis-specific histone (H1t) and the transition proteins (TP1 and TP2) during normal human spermatogenesis. H1t mRNA could only be detected in the cytoplasm of mid and late pachytene spermatocytes. Concomitant with the onset of H1t transcription, the H1t protein appeared in the nuclei of pachytene spermatocytes and remain as a nuclear protein constituent up to step 5 spermatids. While transition protein 1 gene TNP-1 mRNA was present in spermatids from step 2 to early step 4, the TP1 protein occured, with temporal delay, in the nuclei of step 3 and step 4 spermatids. The TP2 protein was observed in the nuclei of spermatids from step 1 to step 5. The transition protein 2 gene TNP-2 mRNA was only detected by reverse transcription-polymerase chain reaction, but not on paraffin sections. These data demonstrate a strong temporal association between H1t gene transcription and synthesis of the H1t protein. Since the TP1 protein appeared with temporal delay we can assume that the corresponding TNP-1 mRNA is translationally delayed.

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Histones are the major protein constituents of the chromatin of eukaryotic cell nuclei. This group of basic proteins is extremely conserved throughout evolution and includes five classes termed H1, H2A, H2B, H3 and H4. In mammals, each of these classes except H4 is subdivided into several subtypes. The most divergent class of histones is the H1 protein family, which consists of seven different subtypes, termed H1.1-H1.5, H1 degree, and H1t. The subtypes H1.2 and H1.4 are found in most somatic cell nuclei, whereas H1 degree is found in several differentiated tissues, and H1t is restricted to mammalian testicular cells. Similarly, core histone subtypes replacing the major forms of H2A, H2B or H3 have been described. Biochemical analysis of protein and RNA from different tissues and cell lines demonstrates varied patterns of expression of individual histone subtype genes. Moreover, antibodies against specific histone subtypes and in situ hybridization with subtype-specific probes indicate that the expression of histone subtype genes is in several cases modulated in a tissue-specific manner. This is particularly evident at the different stages of spermatogenesis when chromatin undergoes substantial reorganization, which finally results in the highly condensed state of chromatin of the mature sperm head.

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The maintenance of a differentiated chondrocyte phenotype is influenced by several factors of which signal transduction of extracellular stimuli through the cell membrane is of major interest. One important group of membrane-bound proteins which are involved in transmembrane signal transduction are ion channels. Human articular chondrocytes were obtained from osteoarthritic femoral condyles. Cells were released from the surrounding matrix and cultivated under standard conditions. We investigated gene expression of 12 members of the TRP ion channel family of freshly prepared (passage 0; P0) and in vitro propagated human articular chondrocytes (passage 2; P2) using conventional and real-time PCR (RT-PCR). In addition, the protein appearance of four TRP channels was demonstrated by immunofluorescence and western blotting. Chondrocyte differentiation was monitored by quantification of collagen type-II, type-I, and aggrecan gene expression. By conventional PCR, 8 channels could be detected, of which some displayed a heterogeneous PCR pattern. RT-PCR quantification revealed that TRPC1 was expressed on the same level in P0 and P2 chondrocytes while gene expression of TRPC3 and TRPC6 was elevated in passage 2 cells. TRPM5, TRPM7, and TRPV1 displayed an enhanced gene expression in freshly isolated chondrocytes. Immunofluorescence signal intensity of all four investigated TRP proteins was consistent with the corresponding gene expression data. In the present study, a correlation between the appearance of some members of the TRP ion channel family and the state of de-differentiation of osteoarthritic articular chondrocytes was shown. A possible direct involvement in the process of chondrocyte de-differentiation has to be investigated in further studies.