Cell- and stage-specific high-level expression of TBP-related factor 2 (TRF2) during mouse spermatogenesis.

Mice lacking the TBP-related factor 2 (TRF2) gene, which is highly expressed in the testis, have a severe defect in spermiogenesis. Here we show that the expression of TRF2 is both cell type- and stage-specific. TRF2 expression was first detected in the late pachytene spermatocytes at stage VIII and increased throughout the subsequent stages. After meiotic divisions, the TRF2 expression declined continuously in round spermatids during progression from stage I to stage V. This observation is consistent with an essential regulatory role of TRF2 in male germ cell differentiation during spermatogenesis.

Spermiogenesis deficiency in mice lacking the Trf2 gene.

The discovery of TATA-binding protein-related factors (TRFs) has suggested alternative mechanisms for gene-specific transcriptional regulation and raised interest in their biological functions. In contrast to recent observations of an embryonic lethal phenotype for TRF2 inactivation in Caenorhabditis elegans and Xenopus laevis, we found that Trf2-deficient mice are viable. However, Trf2-/- mice are sterile because of a severe defect in spermiogenesis. Postmeiotic round spermatids advance at most to step 7 of differentiation but fail to progress to the elongated form, and gene-specific transcription deficiencies were identified. We speculate that mammals may have evolved more specialized TRF2 functions in the testis that involve transcriptional regulation of genes essential for spermiogenesis.

Irradiation selectively inhibits expression from the androgen-dependent Pem homeobox gene promoter in sertoli cells.

How radiation blocks spermatogenesis in certain strains of rats, such as LBNF(1), is not known. Because the block depends on androgen, we propose that androgen affects Sertoli cell function in irradiated LBNF(1) rats, resulting in the failure of spermatogonial differentiation. To begin to identify genes that may participate in this irradiation-induced blockade of spermatogenesis, we investigated the expression of several Sertoli genes in response to irradiation. The expression of the PEM: homeobox gene from its androgen-dependent Sertoli-specific proximal promoter (Pp) was dramatically reduced more than 100-fold in response to irradiation. In contrast, most other genes and gene products reported to be localized to the Sertoli cell, including FSH receptor (FSHR), androgen receptor (AR), SGP1, and the transcription factor CREB, did not exhibit significant changes in expression, whereas transferrin messenger RNA (mRNA) expression dramatically increased in response to irradiation. Irradiation also decreased Pp-driven PEM: mRNA levels in mouse testes (approximately 10-fold), although higher doses of irradiation than in rats were required to inhibit PEM: gene expression in testes of mice, consistent with their greater radioresistance. The decrease in Pem gene expression in mouse testis was also selective, as the expression of CREB, GATA-1, and SGP1 were little affected by irradiation. We conclude that the dramatic irradiation-triggered reduction of Pem expression in Sertoli cells is a conserved response that may be a marker for functional changes in response to irradiation.

Naltrexone for alcohol dependence: a randomized controlled trial.

AIM: We examined the efficacy of naltrexone (an opioid antagonist) for alcohol dependence in a sample of alcohol-dependent men. DESIGN: A 12-week randomized placebo-controlled clinical trial. SETTING: The outpatient clinic of a combined war veteran and general teaching hospital in Melbourne, Australia. PARTICIPANTS: Male alcohol-dependent subjects recruited from the community and from veteran groups. INTERVENTION: Alcohol-dependent subjects were treated with 50 mg of naltrexone or placebo daily for 12 weeks. Both treatment groups attended a weekly education support group. Subjects were assessed weekly. MEASUREMENTS: Primary study outcomes were the maintenance of abstinence and relapse to drinking. FINDINGS: Fifty-five subjects were randomized to naltrexone and 56 to placebo. Forty subjects did not complete 12 weeks of therapy (17 naltrexone, 23 placebo). In the intention-to-treat sample (N = 111) fewer naltrexone treated subjects relapsed (p = 0.001). Among patients who completed the 12-week trial, naltrexone reduced the consumption of alcohol. Naltrexone was well tolerated and there were few adverse experiences. CONCLUSIONS: These findings demonstrate that naltrexone is effective in preventing relapse to drinking in the setting of limited psychosocial treatment. Further studies should examine the duration of treatment needed to maintain the effect long term.

Interleukin-6 regulation of kappa opioid receptor gene expression in primary sertoli cells.

Three classes of opioid receptors--mu, delta, and kappa--mediate physiological and pharmacological functions of the endogenous opioid peptides and exogenous opioid compounds in the central nervous system (CNS), as well as in peripheral tissues including the immune system. Using reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we show that freshly isolated and highly purified somatic (Sertoli and Leydig) and specific germ (spermatogonia, pachytene spermatocytes, round, and elongating spermatids) cells of the rat testis differentially express the mRNAs for these opioid receptor genes. Furthermore, to identify a functional mechanism for cytokine regulation of testicular opioid receptor gene expression, we employed primary Sertoli cells as a model system. In a semiquantitative PCR analysis using the S16 ribosomal RNA gene as an internal control, we show that interleukin-6 reduces kappa opioid receptor mRNA levels from 6 to 24 h of treatment in primary Sertoli cells. This regulation requires new RNA and protein synthesis and is partially mediated by the protein kinase A pathway. These findings are consistent with a role for the cytokine and opioid signaling pathways in Sertoli cellular function and the interaction that exists between the opioid and the immune systems in the CNS.

Dihydrotestosterone as a selective cellular/nuclear localization vector for anti-gene peptide nucleic acid in prostatic carcinoma cells.

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA that, if allowed to enter the cell, bind to the complementary polynucleotide sequence and inhibit DNA transcription and mRNA translation. Although PNAs have a very limited ability in penetrating nuclei of living cells, there are indications that covalent linkage of the PNA to appropriate vectors, e.g., a nuclear localization signal, permits access to the genome. Here we test the ability of dihydrotestosterone (T) covalently linked to PNA to act as a vector for targeting c-myc DNA to prostatic cancer cell nuclei. LNCaP cells, which express the androgen receptor gene, and DU145 cells, in which the androgen receptor gene is silent, offer a model to test this biologically active hormone as a cell-specific vector. T vector was covalently linked to the NH2-terminal position of a PNA complementary to a unique sequence of c-myc oncogene (PNAmyc-T). To localize PNAmyc-T and vector-free PNA within the cells, a rhodamine (R) group was attached at the COOH-terminal position (PNAmyc-R, PNAmyc-TR); cellular uptake was monitored by confocal fluorescence microscopy. PNAmyc-R was detected only in the cytoplasm of both prostatic cell lines, whereas PNAmyc-TR was localized in nuclei as well as in cytoplasm of LNCaP cells. In contrast, PNAmyc-TR uptake in DU145 cells was minimal and exclusively cytoplasmic. In LNCaP cells, MYC protein remained unchanged by exposure to vector-free PNAmyc, whereas a significant and persistent decrease was induced by PNAmyc-T. In DU145 cells, MYC expression was unaltered by PNAmyc with or without the T vector. Our data show that the T vector facilitates cell-selective nuclear localization of PNA and its consequent inhibition of c-myc expression. These findings suggest a strategy for targeting of cell-specific anti-gene therapy in prostatic carcinoma.

Function of stem cell factor as a survival factor of spermatogonia and localization of messenger ribonucleic acid in the rat seminiferous epithelium.

To address the possibility that stem cell factor (SCF) is a paracrine regulator of germ cell development in the adult rat testis, stage-specific distribution of SCF messenger RNA (mRNA) was investigated with Northern blot and in situ hybridization analyses. The highest levels of SCF mRNA were found in stages II-VI of the rat seminiferous epithelial cycle, whereas the lowest levels were in stages VII-VIII. Intermediate levels of SCF mRNA were detected in stages IX-XIV-I of the cycle. The expression of the SCF gene was found to be developmentally regulated, and the expression pattern followed the process of Sertoli cell proliferation and differentiation during postnatal life. The effect of mouse recombinant SCF on spermatogonial DNA synthesis was studied using an in vitro tissue culture system for stage-defined seminiferous tubules. A significant increase in DNA synthesis in spermatogonia could be detected when tubule segments from stage XII were cultured in the presence of 100 ng/ml SCF for 48 h (P < 0.05) and 72 h (P < 0.01). This observation was further confirmed with autoradiographic analyses; almost a 100-fold increase in thymidine incorporation in the SCF-treated (100 ng/ml) tubule segments was observed compared with that in untreated samples. The results of the present study suggest that SCF is a Sertoli cell-produced paracrine regulator and acts as a survival factor for spermatogonia in the adult rat seminiferous epithelium in a stage-specific manner.

Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors.

Gonadal development and differentiation is dependent in part on GH, as GH deficiency has been implicated as a cause of lowered fertility and spermatogenic cessation in humans and some biological models. In this study, we demonstrate that GH receptor messenger RNA (mRNA) is preferentially expressed in progenitor Leydig cells (PLCs) isolated and purified from 21-day-old rats. GH induces significant increases in the levels of steroidogenic acute regulatory protein (StAR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) expression, and androgen production in PLCs. Additionally, the cytokine interferon-gamma (IFNgamma) markedly inhibits GH-stimulated StAR mRNA and protein levels. When cells are cultured with both GH and IFNgamma, IFNgamma decreases the stimulating effect of GH on androgen production. Treatment of PLCs with cycloheximide does not prevent the GH-induced StAR mRNA, indicating that GH induction of StAR transcripts does not require de novo protein synthesis. In contrast, the induction of 3beta-HSD mRNA by GH is altered by cycloheximide treatment. H7, a serine/threonine kinase inhibitor, completely abrogates the increases in StAR mRNA by GH, whereas the tyrosine kinase inhibitor genistein does not. Moreover, GH further enhances StAR and 3beta-HSD mRNA expression in isolated adult rat Leydig cells despite their increased basal expression subsequent to maturational acquisition of these steroidogenic components. These data provide the first demonstration of the direct effects of GH on testicular steroidogenesis during progenitor Leydig cell differentiation.

Kzf1 - a novel KRAB zinc finger protein encoding gene expressed during rat spermatogenesis.

Two novel KRAB (Krüppel associated box) type zinc finger protein encoding cDNAs, named Kzf1 and Kzf2 (Kzf for KRAB zinc finger), were identified by screening of a rat embryonic brain cDNA library with a human ZNF91 KRAB probe. Kzf1 and Kzf2 encode proteins with an amino-terminal KRAB domain and a carboxy-terminal zinc finger cluster containing 9 and 13 zinc finger units, respectively. While Kzf2 appears to be ubiquitously expressed, Kzf1 is preferentially expressed in the testis. Within the testis, Kzf1 mRNA is restricted to germ cells. The Kzf1 protein exhibits DNA binding activity and its KRAB domain can function as a repressor module in transcription. Using somatic cell hybrid analysis, the Kzf1 gene was mapped to chromosome 6.

Functional role for the c-Abl tyrosine kinase in meiosis I.

The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. The DNA-dependent protein kinase (DNA-PK) and the ataxia telangiectasia mutated (ATM) gene product, effectors in the DNA damage response, contribute to the induction of c-Abl activity. The present study demonstrates that c-Abl is expressed in mouse and rat testes, and predominantly in pachytene spermatocytes of meiosis I. The results also demonstrate that c-Abl interacts directly with meiotic chromosomes. In concert with a requirement for c-Abl at the pachytene stage, we show that, in contrast to wild-type mice, testes from Abl-/- mice exhibit defects in spermatogenesis. These findings provide the first demonstration that c-Abl plays a functional role in meiosis.

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression.

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.

Lactogenic hormone-inducible phosphorylation and gamma-activated site-binding activities of Stat5b in primary rat Leydig cells and MA-10 mouse Leydig tumor cells.

The signal transducer and activator of transcription Stat5b has been implicated in signal transduction pathways for a number of cytokines and growth factors, including GH and PRL. Although these lactogenic hormones have the potential to enhance gonadotropin-induced steroidogenesis, the role of GH and PRL in the testis has long been and remains the subject of controversy. In this report we provide, to our knowledge, the first evidence of Stat5b protein expression in the testis and characterize the activation of Stat5b by these lactogenic hormones in primary rat progenitor, immature and adult Leydig cells, and mouse MA-10 Leydig tumor cells. In MA-10 cells, both GH and PRL mediate tyrosine phosphorylation of Janus kinase (JAK) 2 and Stat5b and induce DNA-binding activity of Stat5b. GH enhances both PIE (PRL-inducible element) and Fc gammaRI gamma-activated sites (GAS), but PRL modulates only PIE GAS. In primary Leydig cells isolated from 18-day-old rats, GH, but not PRL, activates cytoplasmic Stat5b and induces the binding of translocated nuclear Stat5b to GAS elements. Although Stat5b protein is expressed in both Percoll- and elutriator-purified adult rat Leydig cells, neither GH nor PRL treatment results in Stat5b-DNA binding. Our studies indicate that the MA-10 cell has the capacity to bind both GH and PRL and provides a useful model system with which to study the distinct testicular roles of these hormones. Moreover, our findings suggest that progenitor and immature Leydig cells are functional targets for GH in the immature rat, suggestive of a role for GH-Stat5b in testicular development. Our data indicate that lactogenic hormone-inducible transcriptional activation may target distinct gene expression in a signaling cascade(s) involving Stat5b but also imply coordinate control by multiple Leydig cell factors.

Testicular leukemia inhibitory factor (LIF) and LIF receptor mediate phosphorylation of signal transducers and activators of transcription (STAT)-3 and STAT-1 and induce c-fos transcription and activator protein-1 activation in rat Sertoli but not germ cells.

Increasing amounts of evidence suggest noninflammatory roles for growth factor and cytokines in development and differentiation. Leukemia inhibitory factor (LIF) belongs to a gp130 pleiotropic family of growth factors that has recently been shown to enhance the survival of rat testicular gonocytes and Sertoli cells. In this study, we show the expression of gp130 and LIF messenger RNAs (mRNAs) in the somatic (the Sertoli and Leydig cells) and specific germ cells (spermatogonia, pachytene, round, and elongated spermatids) of rodent testis, suggestive of cell-specific LIF-mediated functions. LIF receptor mRNA was demonstrated in rat somatic cells, rat elongating spermatids, and all of the mouse germ cells. In addition, we characterized the effects of LIF on the signal transducers and activators of transcription (STAT)-3 and STAT-1, c-fos gene expression, and activator protein-1 regulation in primary rat Sertoli cells. Electrophoretic mobility shift assay and Western blot analysis demonstrated that LIF translocates STAT-3 (and to a lesser extent STAT-1) transcription factor(s) to the nucleus within 2 min of exposure in a tyrosine but not serine/threonine phosphorylation-dependent pathway. Quantitative solution hybridization analysis revealed a transient increase in c-fos mRNA levels by 20-fold following 30-45 min of LIF treatment, an effect that was inhibited by the tyrosine, as well as serine/threonine kinase inhibitors, genistein, and H7. Subsequently, LIF treatment of the Sertoli cells increased nuclear activator protein-1 binding proteins at 2 h after its addition, an effect that was also sensitive to genistein and H7 pretreatments. In contrast, LIF treatment of primary rat germ cells did not alter c-fos mRNA levels. Species specificity in the expression of LIF receptor as well as ligand binding may play a role in LIF signaling in these germ cells. Thus, using a primary Sertoli cell model, we demonstrated that the testicular LIF signaling pathway is contingent on the phosphorylation of latent transcription factors. Our data are consistent with LIF-mediated signaling events involving both somatic and germ cells during spermatogenesis.

Transcriptional regulation of Sertoli cell immediate early genes by interleukin-6 and interferon-gamma is mediated through phosphorylation of STAT-3 and STAT-1 proteins.

The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-gamma induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-gamma increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-gamma, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3-6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription.

Differential activation of signal transducer and activator of transcription (STAT)-3 and STAT-1 transcription factors and c-fos messenger ribonucleic acid by interleukin-6 and interferon-gamma in Sertoli cells.

The regulation of expression of the immediate early gene c-fos messenger RNA (mRNA) by interleukin-6 (IL-6) and interferon-gamma (IFN gamma) was investigated using primary Sertoli cells established from immature rat testis. Using electrophoretic mobility shift assay, we show that IL-6 and IFN gamma predominantly activate signal transducers and activators of transcription-3 and -1 proteins after 15 min of treatment, respectively, whereas solution hybridization and Northern blot analyses show a differential induction of the c-fos mRNA by the two cytokines. IL-6 stimulated c-fos mRNA levels by 6-fold after 45 min; these levels returned to control values by 12 h of treatment. In contrast, IFN gamma increased c-fos message 2-fold transiently, with RNA at the control level by 2 h after treatment. These cytokine-dependent inductions of c-fos mRNA levels were not affected by cyclohexamide, but were diminished by actinomycin D pretreatments, consistent with regulation at the transcriptional level. The differential activation of this testicular c-fos gene thus suggests multiple mechanisms and/or pathways of cytokine regulation in Sertoli cells.

Lesion location and poststroke depression.

This study examined whether stroke lesions involving left hemisphere prefrontal or basal ganglia structures are associated with poststroke depression. A consecutive series of first-ever stroke patients with single small lesions on CT scan were examined for the presence and severity of poststroke depressive disorder. Lesions involving left prefrontal or basal ganglia structures were compared with other left hemisphere lesions and all right hemisphere lesions. Forty-one patients were examined. Patients with lesions involving left hemisphere prefrontal or basal ganglia structures had a higher frequency of depressive disorder (9/12; 75%) than other left hemisphere lesions (1/12; 8%) or those with right hemisphere lesions (5/17; 29%), P = 0.002. These findings suggest that damage to neural pathways within left hemisphere prefrontal or basal ganglia structures is associated with depressed mood following stroke.

Neuroleptic malignant syndrome and risperidone: a case report.

OBJECTIVE: To describe a case of neuroleptic malignant syndrome associated with risperidone. CLINICAL PICTURE: An elderly patient with bipolar affective disorder presented with neuroleptic malignant syndrome and relapse of hypomania after commencing risperidone. TREATMENT: Risperidone was ceased and the patient monitored closely. OUTCOME: The symptoms of neuroleptic malignant syndrome were resolved. CONCLUSION: To our knowledge this is the first such case reported, and suggests that risperidone, like other neuroleptics, is associated with neuroleptic malignant syndrome.

Invasion of the CAG triplet repeats by a complementary peptide nucleic acid inhibits transcription of the androgen receptor and TATA-binding protein genes and correlates with refolding of an active nucleosome containing a unique AR gene sequence.

The DNA sequence of the genes for the androgen receptor (AR) and TATA-binding protein (TBP), like many other genes encoding transcription factors, contains a series of tandem CAG repeats. Here we explore the capacity of complementary peptide nucleic acids (PNAs) to invade the CAG triplets of the AR and TBP genes in human prostatic cancer cells and show that the PNAs readily entered the nuclei of lysolecithin-permeabilized cells and effectively inhibited sense transcription of unique AR and TBP DNA sequences downstream of the site of PNA.DNA hybridization, but not upstream of that site. These PNAs had little or no effect on transcription of the c-myc gene, which lacks a CAG triplet domain. Conversely, a PNA complementary to a unique sequence of the c-myc gene did not inhibit transcription of the AR or TBP genes but did inhibit c-myc transcription. Comparisons of PNA effects on sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA.DNA hybridization is impaired in both directions. Suppression of the AR gene results in refolding of a transcriptionally active nucleosome containing a unique 17-mer AR DNA sequence.

Lesion characteristics and depressed mood in the stroke data bank study.

This study examined the relationship between post-stroke lesion size and location and depressed mood by using data from the multicenter National Stroke Data Bank. For in patients with first-ever cerebral infarction, lesions were characterized by location and size from CT scans. Forty-seven (24%) of the 193 patients studied were depressed. In the complete sample, neither lesion size nor location was associated with depression. However, among patients with comparable small-sized lesions (n = 124), depression was more frequent among those with left hemisphere stroke than those with right hemisphere stroke (31% vs. 16%; P = 0.04). Among patients with larger lesions, brain edema was common and may have obscured lateralized findings. Different biogenic amine neurotransmitter responses to right and left hemisphere brain injury may underlie this mood asymmetry.