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TFIID, comprising the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleation in the assembly of the RNA polymerase II preinitiation complexes on protein-encoding genes. TAFs are shared among other transcription regulatory complexes (e.g., SAGA, TBP-free TAF-containing complex [TFTC], STAGA, and PCAF/GCN5). Human TAF10, a subunit of both TFIID and TFTC, has three histone fold-containing interaction partners: TAF3, TAF8, and SPT7Like (SPT7L). In human cells, exogenously expressed TAF10 remains rather cytoplasmic and leptomycin B does not affect this localization. By using fluorescent fusion proteins, we show that TAF10 does not have an intrinsic nuclear localization signal (NLS) and needs one of its three interaction partners to be transported into the nucleus. When the NLS sequences of either TAF8 or SPT7L are mutated, TAF10 remains cytoplasmic, but a heterologous NLS can drive TAF10 into the nucleus. Experiments using fluorescence recovery after photobleaching show that TAF10 does not associate with any cytoplasmic partner but that once transported into the nucleus it binds to nuclear structures. TAF10 binding to importin beta in vitro is dependent on the coexpression of either TAF8 or TAF3, but not SPT7L. The cytoplasmic-nuclear transport of TAF10 is naturally observed during the differentiation of adult male germ cells. Thus, here we describe a novel role of the three mammalian interacting partners in the nuclear localization of TAF10, and our data suggest that a complex network of regulated cytoplasmic associations may exist among these factors and that this network is important for the composition of different TFIID and TFTC-type complexes in the nucleus.

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.

Using an anti-Fos family member antiserum, we previously described, in the testis of Rana esculenta, the presence of a nuclear 43-kDa protein that we hypothesized to be Fra-1. Using an antiserum against Fra-1, we here report on Fra-1 expression, localization, and putative activity in the R. esculenta testis during the annual reproductive cycle. Western blot analysis confirms that the nuclear 43-kDa protein is Fra-1. Immunocytochemistry demonstrates Fra-1 in peritubular myoid cells (PMC), efferent ducts, and blood vessels. We present, for the first time for a vertebrate, experimental evidence that the expression of Fra-1 in PMC is related to its activity during sperm transport from the tubular compartment to the efferent ducts.

Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding protein family and its associated cofactors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator cAMP response element modulator (CREM) represents an established paradigm. Somatic cell activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CREB-binding protein. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, activator of CREM in the testis (ACT), which confers a powerful, phosphorylation-independent activation capacity. The function of ACT was found to be regulated by the testis-specific kinesin KIF17b. Here we discuss some aspects of the testis-specific transcription machinery, whose function is essential for the process of spermatogenesis.

MSJ-1 is member of the DnaJ/heat shock protein (Hsp) 40 chaperone protein family. It is present in mouse testis and spinal cord. In particular, MSJ-1 is localized in post-meiotic cells and in motoneurones of the ventral horns. To assess whether the role of this protein is evolutionarily conserved, we have investigated if msj-1 gene is expressed in the frog, Rana esculenta. Using reverse transcription-polymerase chain reaction (RT-PCR), a msj-1-like transcript was detected in testis, brain, and spinal cord. Homology ranging from 42.3 to 46.0% was found as compared with the mammalian counterparts. Muscle did not show any signal. By Western blot analysis, a signal of the predicted size of 30 kDa was evidenced in testis, brain, and spinal cord but not in ovary, heart, liver, kidney, and muscle. MSJ-1 fluctuations in the testis reveal that it appeared in concomitance with post-meiotic events during the annual sexual cycle, as shown in a previous study. The protein is localized in spermatids and is still retained in mature spermatozoa, where it has perinuclear and centriolar localization. MSJ-1 levels did not change in brain and spinal cord. Furthermore, in the brain MSJ-1 was mainly present in diencephalon and mesencephalon, while in spinal cord MSJ-1 was localized into several motoneurones of the cervical and thoracic tract. A putative role in vesicle trafficking is briefly discussed., (Copyright 2004 Wiley-Liss, Inc.)

The differentiation of male germ cell requires spermatogenic stage and cell-specific gene expression that is achieved by unique chromatin remodelling, transcriptional control, and the expression of testis-specific genes or isoforms. Specialized transcription complexes that coordinate the differentiation programme of spermatogenesis have been found in germ cells, which display specific differences in the components of the general transcription machinery. The TATA-binding (TBP) protein family and its associated co-factors, for example, show upregulated expression in testis. In this physiological context, transcriptional control mediated by the activator CREM represents an established paradigm. In somatic cells, activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP. In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT, which confers a powerful, phosphorylation-independent activation capacity. The function of ACT is regulated by a testis-specific kinesin, KIF17b. This study discusses some aspects of the testis-specific transcription machinery, the function of which is essential for the process of spermatogenesis.

The role of Fos proteins in the regulation of germ cell progression during spermatogenesis has been studied in the frog, Rana esculenta. A peculiarity of this animal model is the finding of Fos in cytoplasmic compartment of primary spermatogonia during the resting period of the annual reproductive cycle. Interestingly, Fos is localized in the nuclear compartment when spermatogenesis resumes. Using Western blot analysis, we show that a 52-kDa Fos protein occurs in testicular cytosolic preparations, whereas two different Fos signals of 43 and 68 kDa are typical of the nuclear compartment. The 68-kDa Fos immunoreactive protein increases in nuclear extracts in concomitance with spermatogonia (SPG) proliferation either during the annual sexual cycle or in experimental animal groups where SPG proliferation was induced by thermal stimulus (24 C). Indeed, an increase in proliferating cell nuclear antigen was detectable after thermal induction of mitotic activity. A decrease in the 52-kDa signal and a concomitant increase in the 68-kDa signal is observed in testes of 24 C treated groups. The use of alkaline phosphatase and alkaline phosphatase inhibitors indicates that the 68-kDa protein is a phosphorylated form. Estrogens, which are able to induce SPG proliferation, are responsible for the appearance of the 43-kDa Fos form in nuclear testicular extracts. In conclusion, our results show, for the first time in a vertebrate species, that storage in the cytoplasm, on the one hand, and appearance as well as phosphorylation of Fos proteins in the nucleus of germ cells, on the other hand, regulate spermatogenesis progression during the seasonal breeding. Moreover, the phosphorylated 68-kDa Fos form may be involved in mechanisms underlying SPG proliferation.