Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

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.

The PTEN tumor suppressor is a negative modulator of androgen receptor transcriptional activity.

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.

Methylseleninic acid, a potent growth inhibitor of synchronized mouse mammary epithelial tumor cells in vitro.

Selenium compounds have been shown to be effective chemopreventive agents in several animal models and in cultured cells in vitro. It has been proposed that compounds able to generate monomethyl Se have an increased potential to inhibit cell growth. To test this hypothesis, methylseleninic acid (MSeA) and other compounds that could generate methylselenol rapidly were compared with Se compounds that do not generate monomethyl Se, using a well-characterized synchronized TM6 mouse mammary epithelial tumor model in vitro. MSeA at a low micromolar concentration inhibited TM6 growth after 10- to 15-min treatment times. Cells resumed growth after 24 hr but remained sensitive to the fresh addition of monomethyl Se-generators. Dimethyl selenide (DMSe), a putative metabolite of methylselenol, was inactive. Cells treated with 5 microM MSeA were arrested in G1. The effects of 5 microM MSeA on gene expression were evaluated using the Atlas mouse cDNA expression array. A 10-min exposure with MSeA caused a 2- to 3-fold change in the expression of three genes: laminin receptor 1 (decreased), integrin beta (decreased), and Egr-1 (increased). The results provide experimental support for the hypothesis that monomethylated forms of Se are the critical effector molecules in Se-mediated growth inhibition in vitro.

Se-methylselenocysteine activates caspase-3 in mouse mammary epithelial tumor cells in vitro.

Se-methylselenocysteine (MSC) inhibits mouse mammary epithelial tumor cell (TM6) growth. When synchronized TM6 cells were exposed to 50 microM MSC, either for 30 minutes or continuous, the 116 kDa poly(ADP-ribose)polymerase (PARP) was cleaved to an 85 kDa fragment indicative of cells undergoing apoptosis. The earliest cleaved PARP appears at 24 hr time point followed by elevated levels of 85 kDa fragment at 34 hr and 48 hr time points when the cells were exposed to continuous treatment with MSC. Results also showed that MSC increased caspase-3 activity at 24 hr time point. In addition, continuous treatment with MSC induced DNA fragmentation at 34 hr and 48 hr time points with caspase-3 gene expression moderately increased at 16 hr and 24 hr time points. Caspase-6 and -8 were also involved in the MSC-induced apoptosis but to a lesser extent. These results suggest that MSC mediates cleavage of PARP and apoptosis by activating one or more caspases in synchronized TM6 cells and the events are dependent on the duration of treatment.

Androgen binding protein levels and FSH binding to testicular membranes in vitamin A deficient rats and during subsequent replenishment with vitamin A.

ABP levels in the testes and epididymides of vitamin A deficient-retinoic acid maintained rats were only 20 and 6% respectively as compared with those in normal rats. The number of FSH receptors in the testes of vitamin A deficient rats, as measured by [125I]ovine FSH binding to isolated testicular membranes, was only 40% of that in the testes of normal rats. Supplementation of retinyl acetate to these vitamin A deficient rats for 16 days resulted in restoration of the number of FSH receptors to normal levels. On the other hand, ABP levels were restored to 32 and 34% only in the testes and epididymides respectively.

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.

Effect of vitamin A deprivation on the mitogenic factor activity in the rat testes.

The seminiferous tubules of rat testes contain a protease and heat sensitive factor capable of stimulating (3H)-thymidine incorporation into quiescent cultures of NIH 3T3 mouse fibroblast cells. This mitogenic factor activity was only 4% in the testes of vitamin A deficient-retinoic acid maintained rats as compared to that of normal rats. Supplementation of retinyl acetate to these vitamin A deficient rats for 4 days resulted in a 56% recovery in the mitogenic factor activity while by day 16, the recovery was 80 percent. Incorporation of (3H)-thymidine into DNA of the seminiferous tubules of vitamin A deficient rats was only 46% of that of normal rats which was restored to normal levels by retinyl acetate supplementation for 24 days.

Rat spermatid basic nuclear protein TP3 is the precursor of protamine 2.

A second protamine, P2, synthesized as a precursor in late spermatids, is present in the sperm of some mammals such as the mouse. On the other hand, the sperm of other mammals such as the rat do not contain the mature P2, although they have the gene for the protein. Here we report that the P2 precursor is synthesized in late spermatids of the rat and is identical to the protein named TP3. TP3 and four other proteins present in the sonication-resistant spermatids of rat (steps 13-19) immunoreact with an antibody to human P2. By microsequencing the amino terminal ends of the immunoreacting bands, we show that TP3 is the primary translational product of the rat P2 gene and that at least two of the other bands are cleavage products of the precursor molecule. The cleavages occur between residues 10 and 11 and between 19 and 20, as is the case in the mouse. Basic nuclear protein extract of rat epididymal sperm also contains a protein that weakly immunoreacts with the antibody. This protein migrates along with the fastest immunoreacting band from late spermatids. By quantitating the amount of the precursor molecules, we conclude that appreciable levels of P2 precursors are present in rat late spermatids (about 9% of total protamines), but the processing appears to be impaired and most of the protein is lost from the chromatin by the spermatozoa stage.

Stage-specific distribution of the spermatid-specific histone 2B in the rat testis.

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.

Increased accessibility of the N-terminus of testis-specific histone TH2B to antibodies in elongating spermatids.

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N-terminus of the testis-specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross-reacted with TH2B because of sequence homology at the N-termini of TH and TH2B. The epitope was localized to the N-terminus of TH2B as trypsin-digested chromatin which lacked the N-terminal tail did not react with anti-TH and preincubating anti-TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10-12 immunoreacted intensely with anti-TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N-terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones.

Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice.

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 approximately 60% 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.