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.

The testicular H1 histone variant, H1t, is synthesized during spermatogenesis in mammalian male germ cells. In situ hybridization and immunohistochemical techniques were used to assign the expression of either the H1t mRNA or the H1t protein to specific cell stages of spermatogenesis. Our results show the presence of the H1t mRNA only in the late and mid-pachytene stages, whereas the protein occurs first in pachytene spermatocytes, and persists until later stages from round up to elongated spermatids.

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.

The mammalian H1 histone gene complement consists of at least seven H1 protein isoforms. These include five S-phase-dependent H1 protein subtypes and two more distantly related proteins, which are expressed upon terminal differentiation (H1o) or during the pachytene stage of spermatogenesis (H1t). In the past, three replication-dependent murine H1 genes plus the H1o and H1t genes have been isolated and characterized. In this report, we describe the sequences of two more H1 genes, and we show that all five murine replication-dependent H1 genes and the H1t gene map to the region A2-3 on Chromosome (Chr) 13. This is in agreement with our previous finding that the human H1 histone gene complement maps to 6p21.3, which corresponds to the A2-3 region on the murine Chr 13. Previous reports have shown that the replication-independent H1o genes map to syntenic regions on Chrs 22 (human H1o) and 15 (murine H1o).

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