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PubMed1982

Preparative electrophoresis of histones in solubilizable polyacrylamide gels.

Histones from the mouse testis have been fractionated on 17% acrylamide gels containing 0.19% Bis-acrylylcystamine (BAC), 25 M urea and 0.4% Lubrol-WX at pH 2.7. Polyacrylamide gels which contain BAC, a cross-linking agent with disulfide bonds, can be solubilized in the presence of 2-mercaptoethanol, at pH 8.3. Polymerization is carried out at 40 degrees C and in the presence of 6.5--7.5% tetramethylethylenediamine (TEMED) at pH 8.3. Gels containing high acrylamide concentration (greater than 15%) are not soluble if polymerized at lower pH or TEMED concentrations. Following electrophoresis, the gels are stained with Coomassie brilliant blue R for the visualization of protein bands. The stained protein bands are excised and adjusted to pH 8.3 prior to their solubilization. The histones are recovered by ion-exchange chromatography and are free of soluble gel components and dye. Two variants of histone H2B (H2B . 1 and H2B . 2) have been isolated at approx. 95% purity using this single purification step.

Amino Acids↗
PubMed1990

Cytotoxic effects of cyclophosphamide in the mouse seminiferous epithelium: DNA flow cytometric and morphometric analysis.

Stage-specific cytotoxicity of cyclophosphamide (CP) in the mouse testis was analyzed by quantitative DNA flow cytometry and morphometry. In five series of experiments, three mice were injected (ip) with either a single dose of CP at 30 mg/kg or a vehicle saline. At 3, 11, and 20 days later, spermatogenic cells from stages II-V, VII-VIII, and IX-XI of the seminiferous epithelial cycle were quantified by flow cytometry and by morphometry. CP killed a part of the differentiating spermatogonia which became apparent at 11 days when the number of panchytene spermatocytes at stages II-V and VII-VIII was decreased. At stages IX-XI, the number of leptotene and zygotene spermatocytes declined at this time point. These damages could be detected by morphometry, while flow cytometry showed only the trend of the difference. At 20 days, both methods revealed a decrease in the number of haploid spermatids at stages VII-VIII (48 and 33% decrease detected by morphometry and flow cytometry, respectively). DNA flow cytometry proved to be a rapid and practical method to detect stage-specific disruption of spermatogenesis, while morphometry was more sensitive.

Animals↗
PubMed1980

Relation of membrane property of microsomes to androgen biosynthesis.

Spin-labelled fatty acids I(12,3) and I(1,14) were incorporated into microsomal membrane of cryptorchid mouse testis and Leydig cell tumor as well as liver. The freedom of motion of spin of I(12,3) was more restricted in testis microsome than in liver. At the lower temperatures, the freedom of motion of spin in the tumor microsomes was similar to that in the testis, but at higher temperature (20-50 degrees C) was much greater. Plotting of the empirical parameter, h0/h-1, calculated by the spectra of I(1,14), against the reciprocal of the absolute temperature clearly showed two inflection points in both liver and testis microsomes, one at 19 decrees C and the other at 30 degrees C. On the other hand, tumor microsomes lacked these break points and permitted spin to move more freely. These results suggest that tumor microsomes contain the increased fluidity. The importance of membrane fluidity in relation to steroid biosynthesis was also discussed.

Androgens↗
PubMed1996

A novel endozepine-like peptide (ELP) is exclusively expressed in male germ cells.

A cDNA clone encoding a novel endozepine-like peptide (ELP) was isolated from mouse testes, sequenced, and its mRNA expression characterized by northern and in situ hybridization. ELP mRNA was found exclusively in the late spermatid stages of spermatogenesis in the testes of sexually mature mice and in no other tissue or cell type examined. It was also expressed in rat, bovine, porcine and sheep testes. Mouse ELP-encoding cDNA was used to construct expression vectors for the production of ELP in bacteria, and the purified bacterial protein used to raise polyclonal antibodies in rats. These antibodies identified the predicted endogenous ELP in extracts of mouse testis and epididymis and in no other tissue. Immunohistochemistry confirmed that the ELP antigen was present only in late spermatids and spermatozoa, particularly within the cytoplasmic droplet which is retained by the mature spermatozoa during their transit into the epididymis. We conclude that ELP is an intracytoplasmic peptide exclusively expressed in post-meiotic spermatozoa and which may be involved in the energy metabolism of the mature sperm.

Amino Acid Sequence↗
PubMed1984

Immunocytochemical localization of a histone H2A variant in the mammalian nucleolar chromatin.

The distribution of protein "A", a minor variant of H2A present in the mouse testis, was studied in the liver and brain nuclei using peroxidase-antiperoxidase technique. The data presented here suggest that nucleolar-associated chromatin is highly enriched in protein "A". Microspectrophotometric measurements corroborate the immunocytochemical data. The regional differentiation in the eukaryotic chromatin, therefore, may involve qualitative changes in the histone composition.

Animals↗
PubMed1991

A zinc finger protein-encoding gene expressed in the post-meiotic phase of spermatogenesis.

Spermatogenesis is the complex series of physiological and morphological changes that occur when spermatogonial stem cells differentiate into mature spermatozoa. Some of these changes are likely to be regulated at the level of transcription. To approach this problem, we have cloned a cDNA from mouse testis, encoding a protein (Zfp-29) with 14 copies of the zinc finger (Zf) motif commonly found in transcriptional regulatory proteins. The expression of this gene, Zfp-29, is restricted to the testis in adult mice, but also occurs during embryonic development. Within the testis, Zfp-29 mRNA is enriched in round spermatids, the earliest post-meiotic cells. Thus, the putative Zfp-29-encoded protein may have a role in regulating the class of genes that are expressed in post-meiotic germ cells.

Amino Acid Sequence↗
PubMed1994

A mouse cDNA encoding a protein with zinc-fingers and a KRAB domain shows similarity to human profilaggrin.

As part of our efforts to understand transcriptional regulation in the vertebrate eye lens, we have isolated a clone that encodes a zinc finger (Zf) protein from a newborn mouse lens cDNA library. Corresponding message for this protein is detectable in the lens, liver, heart, kidney, spleen and brain of newborn mice. A longer cDNA containing the complete ORF for the same protein was isolated from an adult mouse testis library. A conceptual translation of the testis cDNA sequence produces a 555-amino-acid (aa) protein that contains nine C-terminal Zf and an N-terminal domain found in a subset of C2H2 Zf, the Krüppel-associated box (KRAB). The aa sequence located between the KRAB domain and the Zf shows an unexpected similarity to human profilaggrin, a protein expressed in differentiating epidermal cells. Sequences that hybridize to this cDNA are detectable in ten other mammalian species.

Amino Acid Sequence↗
PubMedDate not supplied

Reversible effects of mercuric chloride on reproductive organs of the male mouse.

Effects of oral administration of mercuric chloride (HgCl2, 1.25 mg/kg) daily for 30 d on the mouse testis, vas deferens, epididymis, and cauda epididymal sperm were investigated. Testis, vas deferens, and epididymis functions were evaluated with respect to sperm count, motility, and viability, and biochemical tests, including succinate dehydrogenase (SDH), adenosine triphosphatase, sialic acid, protein, cholesterol, and glycogen levels in these tissue. Sperm morphology and sperm nuclear integrity were evaluated with standard staining methods. Treatment did not affect whole body and tissue weights. Sperm parameters and fertility were reduced by HgCl2 and most of the biochemical parameters declined. Morphologic histologic alterations were also observed in the tissues studied. All parameters partially recovered after withdrawal of HgCl2 for 45 d.

Animals↗
PubMed1993

[35S]cysteamine: facile synthesis, in vivo biokinetics, and subcellular distribution.

Whereas chemical radioprotection against external beams of ionizing radiation is well studied in radiobiology, the aspects relating to tissue incorporated radionuclides have received little attention. The increased use of radionuclides in diagnostic and therapeutic nuclear medicine, as well as the presence of both manmade and natural radioactivity in the environment, indeed call for such investigations. Our ongoing work on a variety of radioprotectors has revealed that cysteamine (MEA), S-2-aminoethylisothio uroniumbromide hydrobromide (AET), and others (e.g. ascorbic acid), protect spermatogonial cells in mouse testis from the effects of chronic irradiation with intratesticularly localized radionuclides. In these experiments, dose modification factors ranging from 2 to 4 and 10 to 14 were obtained using spermhead survival and induction of spermhead abnormalities, respectively, as the biological end points. Similar experiments were carried out by changing the mode of administration of cysteamine to oral intubation. In these studies a dose modification factor of approximately 3 was observed in the spermhead survival assay. In an effort to understand the protection offered by MEA, the present work describes a one-pot synthesis of high specific activity [35S]cysteamine from elemental [35S]sulphur and its use in determining the biokinetics and biodistribution of MEA following intratesticular (i.t.) or oral administration in mice.

Administration, Oral↗
PubMed2003

Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells.

We cloned a full-length cDNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) including exon Ib from rat and mouse testis. The nuclear signal sequence of the N terminal of rat nuclear PHGPx possessed a different sequence from that previously reported for rat sperm nuclei GPx (SnGPx). Expression of this PHGPx-YFP (yellow fluorescent protein) fusion protein including a novel nuclear signal sequence was exclusively localized in nucleolus; although YFPs fused with only a novel nuclear signal sequence were distributed in the whole nucleus, indicating that preferential translocation of nucleolar PHGPx into nucleoli was required for the nuclear signal sequence and internal sequence of PHGPx. Low level expression of nucleolar PHGPx was detected in several tissues, but the expression of nucleolar PHGPx was extensively high in testis. Immunohistochemical analysis with anti-nucleolar PHGPx indicated that expression of nucleolar PHGPx was observed in the nucleoli in the spermatogonia, spermatocyte, and spermatid. Overexpression of 34kDa nucleolar PHGPx in RBL2H3 cells significantly suppressed cell death induced by actinomycin D and doxorubicin that induced damage in the nucleolus. These results indicated that nucleolar PHGPx plays an important role in prevention of nucleolus from damage in mammalian cells.

Amino Acid Sequence↗
PubMed2005

Altered bioavailability of testosterone in androgen-binding protein-transgenic mice.

Serum and intra-testicular total and free testosterone levels in different age groups of mice (7-360-day-old) were analyzed by radioimmunoassay (RIA) in age-matched wild type (WT)-control and in transgenic mice homozygous to rat androgen-binding protein (ABP-TG), in order to identify possible causes of increased pre-pubertal germ cell apoptosis, spermatogenetic defect and reduced fertility seen in ABP-TG mice. Total intra-testicular testosterone levels in the pre-pubertal ABP-TG (7, 14, 21 and 30-day-old) mice were significantly lower than those in age-matched WT-controls. After puberty (60 days and older) the total intra-testicular testosterone levels were higher than those in age-matched WT-controls and increased gradually, peaking on day 180. Serum total testosterone levels in ABP-TG mice did not differ from those in WT-control until day 30. However, a significant increase in the level of serum total testosterone was observed from day 60. Serum and intra-testicular free testosterone levels were significantly lower in 30, 120, 180 and 360-day-old ABP-TG mice than in age-matched WT-controls. Immunohistochemistry for the cholesterol side-chain cleavage (cytochrome P450) enzyme and quantitative real-time RT-PCR analysis of mRNAs for androgen receptor and for enzymes related to steroidogenesis did not show any changes in 30-day-old ABP-TG mice, indicating that the rates of steroidogenesis and utilization were not altered. Human chorionic gonadotrophin (hCG) administration to adult ABP-TG mice increased the intra-testicular total and free testosterone as well as total germ cell counts. We conclude that the presence of greater than physiological concentration of ABP in the mouse testis alters the ratio of free/bound testosterone, and thereby decreases the availability of free testosterone. As a result, a heightened wave of germ cell apoptosis during the pre-pubertal period followed by a reduction in germ cell numbers and reduced fertility is seen in these mice.

Androgen-Binding Protein↗
PubMed2006

Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor.

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.

Animals↗
PubMed2006

cis-requirement for the maintenance of round spermatid-specific transcription.

Maintenance of strict developmental stage- and cell type-specific gene expression is critical for the progression of spermatogenesis. However, the mechanisms which sustain the spatiotemporal order of gene transcription within the seminiferous epithelium are poorly understood. Previous work has established that the proximal promoter of the mouse SP-10 gene was sufficient to maintain round spermatid-specific expression (Reddi, P.P., Shore, A.N., Shapiro, J.A., Anderson, A., Stoler, M.H., Acharya, K.K., 2003b. Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells. Dev. Biol. 262, 173-182). The present study addressed the cis-requirement for this regulation and sought to identify the cognate transcription factor(s). We found that mutation of two 5'-ACACAC motifs (at -172 and -160) within the -186/+28 SP-10 promoter led to premature and indiscriminate expression of a reporter gene in the seminiferous epithelium of transgenic mice, whereas the wild-type -186/+28 promoter retained spermatid specificity. Neither promoter showed ectopic expression in the somatic tissues. Expression cloning using the -186/-148 portion of the promoter yielded transcriptional repressors TDP-43 and Puralpha of which TDP-43 required the complementary 5'-GTGTGT elements located on the opposite strand for binding in vitro. Further, Northern analysis and immunohistochemistry of mouse testis showed the presence of TDP-43 in cell-types where the SP-10 gene remains repressed. Taken together, our results demonstrate that 5'-GTGTGT motifs on the complementary strand are required to prevent premature expression of SP-10 during spermatogenesis and implicate TDP-43 as the putative regulatory factor. The study also implied that additional level(s) of regulation keep the SP-10 gene silent in the somatic tissues.

Amino Acid Motifs↗
PubMed2004

Human DDX3Y, the Y-encoded isoform of RNA helicase DDX3, rescues a hamster temperature-sensitive ET24 mutant cell line with a DDX3X mutation.

We investigated the function of DDX3Y, the Y chromosome AZFa region encoding a putative DEAD-box RNA helicase protein, the loss of which results in oligozoospermia or azoospermia in humans. The human DDX3Y amino acid sequence is similar to that of the X chromosome gene DDX3X (91.7% homology). Here we report that human Y- and X-encoded DEAD box RNA helicase proteins DDX3Y and DDX3X are interchangeable and have an essential function: both proteins rescued a temperature-sensitive mutant hamster cell line (tsET24) that was otherwise incapable of growth at a nonpermissive temperature. Mouse homologues Ddx3y and D1Pas1-PL10 also rescued the mutant cell line at a nonpermissive temperature. In situ hybridization revealed that Ddx3x mRNA was expressed in almost every cell in mouse testis, suggesting that Ddx3x is involved in spermatogenesis. A comparative study of DDX3X and DDX3Y was performed to determine the significance of DDX3Y for cell growth and spermatogenesis. Both DDX3X and DDX3Y promoter DNAs produced a similar degree of transcription in vivo, whereas deletion studies of the promoter DNAs indicated that these genes are differentially regulated. DDX3Y, similar to DDX3X, shuttles between the nucleus and cytoplasm in a crm1-dependent manner.

Active Transport, Cell Nucleus↗
PubMed2003

Differential localization of ADAM1a and ADAM1b in the endoplasmic reticulum of testicular germ cells and on the surface of epididymal sperm.

Although fertilin is a heterodimeric complex between ADAM1 (A Disintegrin And Metalloprotease 1, fertilin alpha) and ADAM2 (fertilin beta) located on the sperm surface, two different ADAM1 isoforms, ADAM1a and ADAM1b, are present in the mouse testis. In this study, we have examined the localization of ADAM1a and ADAM1b in testicular germ cells and epididymal sperm. ADAM1a was restrictedly present within the endoplasmic reticulum of germ cells, whereas epididymal sperm contained only ADAM1b on the cell surface. The precursors of ADAM1a and ADAM1b formed a heterodimeric complex with that of ADAM2 in the endoplasmic reticulum of germ cells. The heterodimeric complex between the mature forms of ADAM1b and ADAM2 was also found on the sperm surface. These data imply the potential roles of ADAM1a and ADAM1b in spermatogenesis and fertilization, respectively.

ADAM Proteins↗
PubMed1988

Beta-adrenergic receptors and stimulatory effects of (-) isoproterenol on testosterone production in fetal mouse Leydig cells.

Beta-adrenergic receptors were characterized in freshly excised fetal mouse testis using the radioiodinated antagonist iodocyanopindolol (ICYP). [125I]-CYP bound to a single class of high affinity sites with a KD value of 42.2 +/- 7.0 pM. Adrenergic agonists competed for ICYP binding sites with the following order of potency: (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine which is typical for a beta 2-adrenergic receptor. A selective beta 2-receptor antagonist ICI 118-551 showed an approximately 200 fold higher affinity than the beta 1-selective compound, betaxolol. The beta-adrenergic agonist (-)isoproterenol did not or slightly affect testosterone production by freshly isolated fetal Leydig cells. The ability of fetal Leydig cells to respond to (-)isoproterenol increased during culture. This change in responsiveness was not accompanied either by modification of the number of binding sites or by change in the binding affinity. Taken together these data suggest that i) the stimulatory effect of (-)isoproterenol on testosterone production by cultured fetal Leydig cells is mediated through beta 2-adrenergic receptors ii), the inability of freshly Leydig cells to respond to catecholamines is probably due to post receptor events.

Animals↗
PubMed2003

The function of Xenopus germ cell nuclear factor (xGCNF) in morphogenetic movements during neurulation.

The germ cell nuclear factor (GCNF, NR6A1) is a nuclear orphan receptor first described in the mouse testis and subsequently identified as an essential transcription factor in vertebrate embryogenesis. Here, we analyze the phenotype of Xenopus embryos after depletion of embryonic GCNF (xEmGCNF) protein using a specific morpholino antisense oligonucleotide. Morphological defects after xEmGCNF knockdown became obvious from neurulation onward. Among the abnormalities observed, defective formation of the neural tube and a short and curved main body axis were the most remarkable traits. Histological analysis, lineage tracing of injected blastomeres, and Keller sandwich explants revealed that xEmGCNF function is required for different patterns of cell intercalation during neurulation and consequently for the sequence of morphogenetic movements leading to formation of the neural tube. Further characterization of the phenotype at the molecular level showed an abnormal distribution of the extracellular matrix protein fibronectin and a reduction in the expression level of the integrin subunits alpha5 and alpha6, the limiting components of the laminin and fibronectin receptors, respectively. We propose integrin-mediated cell-matrix interaction as a process that requires xEmGCNF function and provides, in concert with cadherins-mediated cell-cell interactions, a molecular basis for morphogenetic cell movements during neurulation.

Animals↗
PubMed1998

The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes.

We have studied the expression of the nuclear orphan receptor GCNF (germ cell nuclear factor) on the mRNA and protein level in pubertal and adult mouse testes. We show by Northern and Western blot analyses and by in situ hybridization that GCNF is expressed in spermatocytes and round spermatids of adult mouse testis suggesting that GCNF may be a transcriptional regulator of spermatogenesis. Since the GCNF protein is accumulated in the XY body of late pachytene spermatocytes, it may be involved in transcriptional inactivation of sex chromosomes.

Animals↗