High accumulation of components of the RNA polymerase II transcription machinery in rodent spermatids

Edward E. Schmidt Ueli Schibler - machinery in rodent spermatids Levels of mRNA and protein encoded by the TATA-binding protein (tbp) gene are shown to increase dramatically during late spermatogenesis in rodents, culminating in a highly testis-enriched expression pattern. Whereas adult spleen and liver contained roughly 0.7 and 2.3 molecules of TBP mRNA per haploid genome-equivalent, respectively, adult testis contained 80-200 molecules of TBP mRNA per haploid genome-equivalent. Comparison of nuclear and cytoplasmic levels of TBP mRNA in liver and testis suggested that nuclear events (transcription or processing) contribute roughly 12-fold, and cytoplasmic events (mRNA stability) roughly 6-fold, to testis-specific overaccumulation. Levels of nuclear TBP protein in testis cells were, on average, 8- and 11-fold higher than those in liver and spleen Spermatogenesis is the process through which diploid stem cells (spermatogonia) differentiate into mature haploid spermatozoa. Major postembryonic steps in mammalian spermatogenesis begin with mitotic proliferation of the spermatogonia. Some of the resultant daughter cells remain as stem cells; the rest differentiate into spermatocytes and undergo meiosis. Each spermatocyte yields four haploid spermatids. During spermiogenesis (the haploid stages of spermatogenesis), the acrosome and flagella are synthesized, histones are replaced by protamines leading to chromatin condensation, and finally, the condensed late spermatid is released from the bulk of its cytoplasm (reviewed in Bellv et al., 1977; Kleene et al., 1983; Leblond and Clermont, 1952; Meistrich, 1989). The net result of this process is a gross morphological, biochemical, structural and genetic transformation that provides one of the most dramatic examples of cell differentiation and specialization. Mature spermatozoa exhibit highly compacted chromatin and are transcriptionally inactive (Monesi, 1964). In mammals, chromatin condensation involves the sequential replacement of somatic histones by transition proteins and protamines (Balhorn et al., 1984; Bellv et al., 1975; Bellv, 1979; Grimes et al., 1977; Kistler et al., 1973; Mayer et al., 1981; Meistrich, 1989). Chromatin condensation is one of the final steps in spermatogenesis. Many of the proteins involved in condensation are synthesized from mRNAs which were, themselves, synthesized several days earlier and stored in a translationally cells, respectively. Overexpression of TBP mRNA in testis began about 20 days after birth and reached a plateau around day 40, corresponding to the developmental emergence of haploid cells. Besides TBP, two other components of the general RNA polymerase II machinery, TFIIB and RNA polymerase II, were also overexpressed in testis. By immunostaining, it was found that TBP and RNA polymerase II were particularly rich in round spermatid nuclei. Our results suggest a molecular explanation for how early spermatids are able to accumulate all of the mRNA necessary for the final week of spermiogenesis. inactive state (Balhorn et al., 1984; Braun et al., 1989; Kleene et al., 1984). For example, transcription of the protamine 1 and 2 genes and the transition protein 1 and 2 genes is detected only in early spermatids, at which time these mRNAs accumulate to very high levels. Translation of these mRNAs occurs several days later (Hecht et al., 1986; Heidaran et al., 1987; Kleene and Flynn, 1987; Kleene et al., 1990; Kleene, 1993). The synthesis of these abundant mRNAs in early spermiogenesis might place stringent demands on the RNA polymerase II (pol II) machinery. We have been interested in the mechanisms regulating overall rates of transcription in metazoan cells. Previous investigations revealed a correlation between cell size and overall transcription rates in various tissues (Schmidt and Schibler, 1995). Because overall transcription rates by all three RNA polymerases appear to be cell size-dependent, we began investigating whether components of the general transcription machinery might be involved in determining cell size-specific transcription rates. An important component of the general transcription machinery is the TATA-binding protein (TBP). TBP functions in promoter recognition and initiation by all three eucaryotic RNA polymerases (reviewed by Hernandez, 1993). In the course of investigating whether TBP might play a role in determining cell size-dependent transcription rates, we noticed that TBP accumulated to much higher levels in testis as compared to all somatic tissues and cell types examined. Within the testis, TBP overexpression was localized to a subset of the germ cells which appeared to be in the early haploid stages. Further analysis suggested that RNA polymerase II was also highly abundant in the nuclei of round spermatids. Our findings suggest that overexpression of the pol II transcription apparatus might be a general property of this stage of spermatogenesis. MATERIALS AND METHODS Animals, sample preparations and immunostaining Male laboratory rats (Lewis) or mice (MORO) were used in all experiments. RNA and nuclear extracts were prepared from tissues and cultured cells as described by Schmidt and Schibler (1995) and Schmidt et al. (1991). For immunostaining, small pieces of decapsulated rat testes were fixed for 2 hours at room temperature in 5% glutaraldehyde/PBS overlaid with heptane. After washing and dehydrating, tissues were embedded in Paraplast supplemented with 0.8% dimethylsulfoxide. Sections attached to 3-aminopropyltriethoxysilane-coated slides were dewaxed in xylene followed by 100, 95, and 80% ethanol. Slides were treated for 20 minutes in 80% methanol, 3% hydrogen peroxide, hydrated, and postfixed in 4% paraformaldehyde/PBS. Sections were preincubated with PBS containing 0.1% Triton X-100, 1% nonfat dry milk (PTM) supplemented with 1% normal goat serum for 1 hour followed by first antibody (1:20) in PTM for 1 hour. Sections were washed five times in PTM and incubated in second antibody (goat-anti-rabbit, or anti-mouse, horseradish peroxidase-conjugated, 1:100 dilutions) in PTM for 1 hour and washed as above. Control sections were incubated with no antibody, with only second antibody-conjugates, or with non-immune rabbit serum followed by anti-rabbit peroxidase conjugate. Staining was with diaminobenzidine. Rabbit-anti-human TBP antiserum (raised against the whole molecule) was a generous gift from A. Hoffmann and R. Roeder; mouse-anti-human TBP monoclonal antibody (undefined epitope) was purchased from Santa Cruz Biotechnology. The mouse-antihuman pol II antibody (CTD epitope) was a generous gift from C. Kedinger. RNase protection assays RNase protection assays were as described by Schmidt and Merrill (1989) and Schmidt and Schibler (1995). Signals were quantitated by liquid scintillation of excised gel bands. Plasmid 6His-pET IId, containing the human TBP cDNA, was a gift from A. Hoffmann and R. Roeder (Hoffmann et al., 1990); a 148-base pair Sau (...truncated)