Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12-13 spermatid nuclei from the testis of Tnp1-null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and Ϸ60% of Tnp1-null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility. The transformation of spermatids into spermatozoa (spermiogenesis) involves the most dramatic changes in chromatin structure and function that occur in any cell type. During the latter part of spermiogenesis, the nucleus elongates, transcription ceases, the histones are almost completely removed, and the chromatin appears as smooth fibers and then becomes highly condensed (1). In many animal and plant species, chromatin condensation is facilitated by the association of highly basic nuclear proteins, the protamines (2). The transition from histone-containing chromatin to the protamine-associated one seems to occur directly in fish and birds (2). However, in mammals (3), small, basic nuclear proteins appear when the histones are displaced and chromatin condensation is initiated; they are referred to as transition nuclear proteins (TPs), because they are subsequently replaced by protamines (3).Although other TPs exist (4), TP1 and TP2 are the predominant ones found in rodent spermatids (5). TP1, a 6.2-kDa protein, consists of Ϸ20% each arginine and lysine and lacks cysteine (6, 7). TP2, a 13-kDa protein, consists of Ϸ10% each arginine and lysine and 5% cysteine (5). TP1 is expressed abundantly in most mammals (6) and is highly conserved, showing cDNA nucleotide and amino acid sequence homologies of 90% across species (8). The TPs are localized exclusively to nuclei of condensing spermatids (3, 9), and in the rat, constitute Ͼ90% of basic chromosomal proteins of these nuclei (10).During human (11) and mouse (12) spermiogenesis, the TPs are replaced by two protamines, protamine 1 (P1) and protamine 2 (P2). Whereas P1 is synthesized as a mature protein, P2 is synthesized as the precursor. In mouse, mature P2 of 63 residues is derive...

A cure for prostate cancer (CaP) will be possible only after a complete understanding of the mechanisms causing this disease to progress from androgen dependence to androgen independence. To carry on a careful characterization of the phenotypes of CaP cell lines before and after acquisition of androgen independence, we used two human CaP LNCaP sublines: LNCaP nan , which is androgen dependent (AD), and LNCaP-HP, which is androgen independent (AI). In AD LNCaP nan cells, dihydrotestosterone (

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

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To investigate whether the tumor suppressor gene PTEN affects the activity of the androgen receptor (AR), we monitored the expression of the apoptotic gene HA-Bax (inserted in an adenovirus where it is driven by the AR-responsive promoter ARR 2 PB) in the presence or absence of dihydrotestosterone, in PTEN (+) or (−) prostate cancer cell lines, infected with an adenovirus containing wild-type PTEN (Av-CMV-PTEN) or a control LacZ-expressing construct. Our results showed that AR transcriptional activity was antagonized by PTEN expression. This antagonism was not cell line dependent, as it was observed in both LNCaP and LAPC-4 cells, or promoter dependent, as it was observed for a reporter gene (HA-Bax) driven by an exogenous androgen-responsive promoter (the ARR 2 PB promoter), and for a native gene (prostate-specific antigen; PSA) driven by an endogenous AR-responsive promoter. Additional experiments performed with viruses containing constitutively active (Adeno-myrAkt) or dominant negative (Adeno-dnAkt) forms of Akt demonstrated that Akt, a protein kinase whose activation is known to be inhibited by PTEN, mediated the observed antagonism between PTEN and AR transcriptional activity. Recently, two putative Akt phosphorylation sites have been identified in the AR sequence. Site-directed mutagenesis was utilized to convert these two serine into alanine residues. The resulting construct, named CMV-AR S213A&S791A was transfected in AR (−) and PTEN (−) PC-3 cells in the presence or absence of Av-CMV-PTEN and of two reporter plasmids (GRE 2 E1b-Luc and PSA P/E-luc) containing the luciferase gene driven by well-characterized androgen responsive promoters. These experiments demonstrated that, similarly to the wild-type molecule, AR S213A&S791A was transcriptionally inhibited by PTEN, suggesting that Akt does not have an effect on AR transcription by direct phosphorylation, but probably by affecting the availability of a downstream molecule whose main mechanism of action is that of modulating AR transcription. The data presented here suggest that loss of PTEN function may facilitate activation of AR signaling and progression to androgen independence in prostate cancer.

Introduction Se-methylselenocysteine (MSC), a naturally occurring selenium compound, is a promising chemopreventive agent against in vivo and in vitro models of carcinogen-induced mouse and rat mammary tumorigenesis. We have demonstrated previously that MSC induces apoptosis after a cell growth arrest in S phase in a mouse mammary epithelial tumor cell model (TM6 cells) in vitro. The present study was designed to examine the involvement of the phosphatidylinositol 3-kinase (PI3-K) pathway in TM6 tumor model in vitro after treatment with MSC.

Background Se-methylselenocysteine (MSC) is a naturally occurring organoselenium compound that inhibits mammary tumorigenesis in laboratory animals and in cell culture models. Previously we have documented that MSC inhibits DNA synthesis, total protein kinase C and cyclin-dependent kinase 2 kinase activities, leading to prolonged S-phase arrest and elevation of growth-arrested DNA damage genes, followed by caspase activation and apoptosis in a synchronized TM6 mouse mammary tumor model. The aim of the present study was to examine the efficacy of MSC against TM6 mouse mammary hyperplastic outgrowth (TM6-HOG) and to determine in vivo targets of MSC in this model system.

Male germ cells contain a number of histone variants, most of which are synthesized during either the mitotic or the meiotic stages of spermatogenesis. A spermatid-specific H2B (ssH2B) variant has been identified in mouse round spermatids that has an additional 12 amino acids at its carboxyl terminus as compared to somatic H2Bs. Until now, the presence of this protein in other mammals has not been known. Northern blot analysis using an oligonucleotide probe complementary to a unique coding region of the C-terminus of mouse ssH2B showed that mRNA encoding this protein was present in isolated rat round spermatids. Furthermore, immunoblot analysis of basic nuclear proteins from round spermatids probed with an anti-ssH2B antiserum that recognizes the novel peptide sequence indicated that the protein was present in round spermatids but not in pachytene spermatocytes. The ssH2B comigrated with H2A.X by acid-urea (2.5 M) PAGE and migrated slightly more slowly than TH2B by acid-urea-Triton PAGE. When seminiferous tubules were microdissected stage-specifically and basic nuclear proteins were immunoblotted, ssH2B was detected in tubule sections of stages II-VI. It reached a maximum level in stages VII-VIII and then decreased to minimal levels in stages XIII-I. Since ssH2B was not detected in pachytene spermatocytes, the stage-specific levels of ssH2B corresponded to the respective steps of spermatids present in the tubules. While ssH2B constituted a relatively small amount (approximately 2%) of total H2B protein in round spermatids, its presence in both the mouse and the rat suggests that its function may be conserved in mammalian spermatogenic cells.