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H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1...

The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1-4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however, the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full-length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1-3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5' of the TATA box and 3' of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5'-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.

Spermatogenesis demonstrates a regulated program of genetic expression driving a complex progression of cellular differentiation. A current challenge is to understand the factors responsible for the many examples of differential gene expression documented during spermatogenesis (1,2). An aspect of male germ-cell differentiation that is fairly well described at the molecular level involves changes in DNA packaging by basic chromosomal proteins. Histones are responsible for the fundamental packaging unit of chromatin, the nucleosome, and early male germ cells contain the standard somatic forms of histones. However, beginning in late spermatogonia, novel histone variants that are unique to the male germ line start to appear. These include the core histone variants TH2A, TH2B, and TH3, as well as the linker histone variant H1t. The variants replace some, though not all, of their somatic-type counterparts, so that by the last third of meiotic prophase the spermatocyte nucleus is substantially enriched for testis-specific histones. This chromatin state persists into round spermatids until the spermatid nuclei elongate and condense (3, 4).

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.

Summary Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.

Cloned gene sequences have been isolated for two testis-specific chromosomal proteins, one of which, histone (H1t), appears during meiosis, whereas the other, transition protein 1 (TP1), appears only during the later steps of spermatid development. Aspects of the regulation of each gene have been examined. In the case of H1t, analysis of its promoter region shows that it contains excellent matches to each of the four sequence homologies identified for the usual somatic H1 variants, so that the factor(s) that restrict H1t expression to spermatocytes remain a mystery. In the case of TP1, a cDNA clone allowed identification of its message by Northern blots as well as by in situ hybridization. The message appears postmeiotically in late round spermatids but is translationally repressed until the spermatid nucleus begins to condense.

Hlt is a testis-specific Hl histone variant associated with meiosis and post-meiotic stages of male germ cell development. We have now made tentative identification of Hlt in humans by a variety of criteria including electrophoretic and extractive properties. While Hlt was readily identified in extracts from normal testes, it was not detectable in extracts from aspermatogenic testes or from placenta. Identification of Hlt in humans confirms the widespread association of this unusual Hl variant with spermatogenesis among mammals ranging from rodents to primates.

The genes for seminal vesicle secretory protein IV and prostate steroid binding protein component 3 of the rat share upstream homologies of which the most striking is a 30 nucleotide sequence located between position −190 and −330 relative to the major transcriptional initiation sites. This sequence does not appear to be a common repetitive element and deserves consideration as a potential site involved in the androgen regulated expression of these genes.

A unique acid-soluble basic protein was detected in the testes of sexually mature rats and a number of other mammalian species. This protein was not found in comparable acid extracts prepared from many other rat organs or from the testes of juvenile rats. It makes its appearance only after spermatids are evident in normally developing testes and is absent from the gonads of male rats rendered artificially cryptorchid for periods of 10 days or longer. This protein was purified extensively and was found to be devoid of phenylalanine, tryptophan, glutamic acid, glutamine, isoleucine, and cyst(e)ine. The specific basic protein from rat testes could not be extracted from epididymal sperm by a variety of procedures. In contrast, certain techniques reproducibly extracted from both epididymal spermatozoa and whole testis a highly basic protein fraction that is strikingly rich both in arginine and cyst(e)ine.