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At least 973 records · Page 54Linked to original sources
PubMed1998

Characterization of domains in mice of calnexin-t, a putative molecular chaperone required in sperm fertility, with use of glutathione S-transferase-fusion proteins.

Calnexin-t (calmegin) is a male germ cell-specific variant of calnexin, a membrane bound-molecular chaperone in the endoplasmic reticulum (ER). Although it is temporally expressed during spermatogenesis, it has recently been shown to be highly involved in sperm fertility. To investigate the biochemical states of calnexin-t during spermatogenesis, we produced a series of glutathione S-transferase-fusion proteins with several specific coding domains of calnexin-t. Immunostaining and 45Ca2+ overlay assays clearly showed that the internal proline-rich repeat region has Ca2+-binding ability and contains an epitope recognized by monoclonal antibody 1C9. Western blot analysis of protein extracts from the testes of 10-, 18-, 26-, and 60-day-old mice revealed only a single 101-kDa protein during testicular development by 1C9. Anti-C, a cytoplasmic domain-specific antibody generated by immunization with recombinant protein, produced the same results, indicating that the 101-kDa form of calnexin-t is prevalent at all stages of spermatogenesis expressing calnexin-t. In paraffin sections of mouse testis, Anti-C stained spermatocytes and spermatids intensely, whereas 1C9 stained spermatocytes only slightly but spermatids intensely, suggesting that the affinity of 1C9 for its epitope is lower in pachytene spermatocytes than in spermatids. Acid phosphatase treatment of the 101-kDa form generated a 93-kDa band that in turn could be recovered to the 101-kDa form by incubation with HeLa cell S100 fraction, indicating that the 101-kDa form is a phosphorylated type of calnexin-t. The sites of phosphorylation were shown to be restricted to the cytoplasmic domain. Our results suggest that the structure of the ER luminal domain of calnexin-t is likely to differ in middle pachytene versus haploid germ cell phases. In addition, the cytoplasmic domain of calnexin-t was shown to be highly phosphorylated immediately after protein synthesis and constitutively phosphorylated during spermatogenesis.

Animals↗
PubMed2000

Association of the mouse infertility factor DAZL1 with actively translating polyribosomes.

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.

Animals↗
PubMed2000

Cloning and characterization of a complementary deoxyribonucleic acid encoding haploid-specific alanine-rich acidic protein located on chromosome-X.

We have isolated a cDNA clone encoding a germ cell-specific protein from an expression cDNA library prepared from the mouse testis using testis-specific polyclonal antibodies. Northern blot analysis showed a transcript of 1.1 kilobases exclusively expressed in haploid germ cells of the testis. Sequence analysis of the cDNA revealed one long open reading frame consisting of 238 deduced amino acids, rich in basic amino acids in the N-terminal one-third that also contained the nuclear localization signal, and rich in acidic amino acids, including two type of acidic alanine-rich repeats, in the rest of the deduced protein. The protein having a molecular weight of approximately 55 kDa and an isoelectric point of pH 4.3-4.7 was also exclusively detected in the testis by Western blot analysis. As the cDNA was located on chromosome-X, Halap-X (haploid-specific alanine-rich acidic protein located on chromosome-X) was proposed for the name of the protein encoded by the cDNA. Immunohistochemical observation revealed that the Halap-X protein was predominantly present in the nucleoplasm of round spermatids but gradually decreased as spermatids matured, followed by the subsequent appearance in the cytoplasm of elongating spermatids. Thus, the Halap-X protein was transferred from the nuclei to the cytoplasm during the spermatid maturation when the chromatin condensation and transformation of the nuclei occurred. The Halap-X may facilitate specific association of nuclear DNA with some basic chromosomal proteins and play important roles in the process of chromatin condensation.

Amino Acid Sequence↗
PubMed2000

Involvement of the D-type cyclins in germ cell proliferation and differentiation in the mouse.

Using immunohistochemistry, the expression of the D-type cyclin proteins was studied in the developing and adult mouse testis. Both during testicular development and in adult testis, cyclin D(1) is expressed only in proliferating gonocytes and spermatogonia, indicating a role for cyclin D(1) in spermatogonial proliferation, in particular during the G(1)/S phase transition. Cyclin D(2) is first expressed at the start of spermatogenesis when gonocytes produce A(1) spermatogonia. In the adult testis, cyclin D(2) is expressed in spermatogonia around stage VIII of the seminiferous epithelium when A(al) spermatogonia differentiate into A(1) spermatogonia and also in spermatocytes and spermatids. To further elucidate the role of cyclin D(2) during spermatogenesis, cyclin D(2) expression was studied in vitamin A-deficient testis. Cyclin D(2) was not expressed in the undifferentiated A spermatogonia in vitamin A-deficient testis but was strongly induced in these cells after the induction of differentiation of most of these cells into A(1) spermatogonia by administration of retinoic acid. Overall, cyclin D(2) seems to play a role at the crucial differentiation step of undifferentiated spermatogonia into A(1) spermatogonia. Cyclin D(3) is expressed in both proliferating and quiescent gonocytes during testis development. Cyclin D(3) expression was found in terminally differentiated Sertoli cells, in Leydig cells, and in spermatogonia in adult testis. Hence, although cyclin D(3) may control G(1)/S transition in spermatogonia, it probably has a different role in Sertoli and Leydig cells. In conclusion, the three D-type cyclins are differentially expressed during spermatogenesis. In spermatogonia, cyclins D(1) and D(3) seem to be involved in cell cycle regulation, whereas cyclin D(2) likely has a role in spermatogonial differentiation.

Animals↗
PubMed2001

Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degrees C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.

Animals↗
PubMed2002

Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells.

To study the mechanism of male germ cell differentiation, testicular germ cells carrying green fluorescent protein (GFP) as a transgene marker were transplanted into infertile mouse testis. Fluorescence-positive seminiferous tubule segments colonized with GFP-labeled donor germ cells were isolated and measured, and differentiated germ cells were analyzed in living squashed preparations. Cell associations in normal stages of the seminiferous epithelial cycle were also studied and used as a reference. Two months after transplantation, the average length of the colonies was 1.3 mm. The cell associations of transplanted colonies were consistent with those of normal stages of the cycle. However, stages of the cycle were not necessarily identical in different colonies. Three months after transplantation, the average length of transplanted colonies was 3.4 mm, and the cell association in every portion of a colony was similar to that of the corresponding stage of the cycle. Even in long fused colonies made by transplantation of a higher concentration of male germ cells, the cell association patterns in various regions of a single colony were similar and consistent with those of some of the normal stages of the cycle. Development of different stages inside the colony was observed by 6 mo after transplantation. These results indicate that the commencement of spermatogonial stem cell differentiation occurs randomly to develop different stages of the cycle in different colonies. Then, each colony shows one single stage of the cycle for a long time, even if it becomes a very large colony or fuses with other colonies. These observations indicate the existence of some kind of synchronization mechanism. By 6 mo, however, normal development of the stages of the cycle appeared in seminiferous tubules.

Animals↗
PubMed2002

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein.

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.

Amino Acid Sequence↗
PubMed1996

Peptide mass fingerprinting of chaperonin-containing TCP-1 (CCT) and copurifying proteins.

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research, CCT isolated from mouse testis lysate sediments at 20S in a sucrose gradient and accounts for about 70% of the total protein in this fraction. We intend to identify all the other proteins that copurify with CCT and to compile a reference profile for future studies. Their identification can be accelerated by a combination of protease digestion, matrix-assisted laser desorption-mass spectrometry, and database matching known as peptide mass fingerprinting. We applied this strategy to 32 polypeptides resolved by 2-dimensional gel electrophoresis, and 23 known proteins and 6 novel proteins were identified. We analyzed isoelectric variants of the CCT subunits and differences in the peptide mass spectra of two CCT theta isoforms indicated a novel posttranslational modification of this subunit.

Amino Acid Sequence↗
PubMed1993

Effects of diatrizoate and iopamidol on spermatogenesis.

RATIONALE AND OBJECTIVES: The biological effects of iodinated contrast media were examined by using spermatogenesis in mouse testis as the experimental model. METHODS: Spermhead survival and abnormality assays were used as the biological end points. Diatrizoate meglumine/diatrizoate sodium and iopamidol were administered intravenously at equal rates and concentrations. Testicular uptake and clearance of these contrast agents were examined by high-performance liquid chromatography techniques. Appropriate mannitol solutions were employed as osmolality controls. RESULTS: Intravenous administration of the contrast agent or its respective mannitol control resulted in approximately a 30% decrease in spermhead count. A dose-related experiment with mannitol demonstrated that the spermhead count decreased rapidly until 600 mOsm/kg was reached, beyond which this decrease was minimal. Clearance of both contrast media was complete in approximately 4 hours. No significant increase in the induction of spermhead abnormalities was observed. CONCLUSION: Osmotic substances, such as iodinated contrast agents, affect the process of spermatogenesis.

Animals↗
PubMed1996

The Atr and Atm protein kinases associate with different sites along meiotically pairing chromosomes.

A number of cell-cycle checkpoint genes have been shown to play important roles in meiosis. We have characterized the human and mouse counterpart of the Schizosaccharomyces pombe Rad3 protein, named Atr (for ataxia-telangiectasia- and rad3-related), and the protein that is mutated in ataxia-telangiectasia, Atm. We demonstrate that ATR mRNA and protein are expressed in human and mouse testis. More detailed analysis of specific cells in seminiferous tubules shows localization of Atr to the nuclei of cells in the process of meiosis I. Using immunoprecipitation and immunoblot analysis, we show that Atr and Atm proteins are approximately 300 and 350 kD relative molecular mass, respectively, and further demonstrate that both proteins have associated protein kinase activity. Further, we demonstrate that Atr and Atm interact directly with meiotic chromosomes and show complementary localization patterns on synapsing chromosomes. Atr is found at sites along unpaired or asynapsed chromosomal axes, whereas Atm is found along synapsed chromosomal axes. This is the first demonstration of a nuclear association of Atr and Atm proteins with meiotic chromosomes and suggests a direct role for these proteins in recognizing and responding to DNA strand interruptions that occur during meiotic recombination.

Animals↗
PubMed1988

The cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation.

In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D0, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D0, 0.43 Gy) than the other phases of the cell cycle (D0, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.

Animals↗
PubMed1991

Processing of the precursor of protamine P2 in mouse. Identification of intermediates by their insolubility in the presence of sodium dodecyl sulfate.

Two basic proteins, protamines P1 and P2, are present in chromatin of mouse spermatozoa. Protamine P1, the less abundant protein in mouse, has a homolog in most mammals, and its synthesis follows a conventional route. In contrast, protamine P2 has been found only in certain other mammals, including humans, and it is synthesized as a precursor nearly twice as long as the mature protein. Processing of this precursor is not yet understood, although it necessarily takes place in elongating spermatids and is likely to play a role in the chromatin condensation occurring in these haploid cells. We have fractionated basic proteins from mouse testis chromatin and have identified six proteins on electrophoretic gels which, like protamines, are insoluble in SDS. All six were also soluble at the same trichloroacetic acid concentration as protamine P2 and were present in chromatin of elongating spermatids. Radioactive labelling patterns acquired by these SDS-insoluble proteins during translation in vitro of testis RNA indicate that the largest represents the precursor of protamine P2, and suggest that the others represent intermediates generated by proteolytic cleavage of the precursor. Results from pulse 3H labelling in vivo were also consistent with the conclusion that a precursor/product relationship exists between these proteins and protamine P2. Conclusions concerning the kinetics of processing have, in addition, been drawn from this data. Hypotheses concerning possible functional roles played by the precursor are presented.

Amino Acid Sequence↗
PubMed1987

Characterization of a testis specific antigen in the mouse.

The partial characterization of a mouse hydrosoluble testis specific protein by crossed immunoelectrophoresis, ion-exchange chromatography and SDS-Page was made. This protein presented a low electrophoretic motility at pH 8.6. It was not adsorbed to DEAE-Sephacel at pH 8.6, but was slightly adsorbed at pH 9.0, while in CM-Sephadex it was readily adsorbed at pH 6.0, indicating the basic nature of this protein. The apparent molecular weight was determined to be of 28 Kd by SDS-Page. The previously reported testis specificity of this protein was corroborated by its absence in a liver extract.

Animals↗
PubMed2002

Can antioxidant diet supplementation protect against age-related mitochondrial damage?

Harman's free radical theory of aging and our electron-microscopic finding of an age-related mitochondrial degeneration in the somatic tissues of the insect Drosophila melanogaster as well as in the fixed postmitotic Leydig and Sertoli cells of the mouse testis led us to propose a mitochondrial theory of aging, according to which metazoan senescence may be linked to oxygen stress-injury to the genome and membranes of the mitochondria of somatic differentiated cells. These concepts attract a great deal of attention, since, according to recent work, the mitochondrial damage caused by reactive oxygen species (ROS) and concomitant decline in ATP synthesis seem to play a key role not only in aging, but also in the fundamental cellular process of apoptosis. Although diet supplementation with antioxidants has not been able to increase consistently the species-characteristic maximum life span, it results in significant extension of the mean life span of laboratory animals. Moreover, diets containing high levels of antioxidants such as vitamins C and E seem able to reduce the risk of suffering age-related immune dysfunctions and arteriosclerosis. Presently, the focus of age-related antioxidant research is on compounds, such as deprenyl, coenzyme Q10, alpha-lipoic acid, and the glutathione-precursors thioproline and N-acetylcysteine, which may be able to neutralize the ROS at their sites of production in the mitochondria. Diet supplementation with these antioxidants may protect the mitochondria against respiration-linked oxygen stress, with preservation of the genomic and structural integrity of these energy-producing organelles and concomitant increase in functional life span.

Aging↗
PubMed1993

Wnt-1-inducing factor-1: a novel G/C box-binding transcription factor regulating the expression of Wnt-1 during neuroectodermal differentiation.

The Wnt-1 proto-oncogene is essential for proper development of the midbrain and is expressed in a spatially and temporally restricted manner during central nervous system development in mice. In vitro, the gene is specifically transcribed during the retinoic acid (RA)-induced neuroectodermal differentiation of the P19 line of embryonal carcinoma cells. The P19 cells differentiate into neurons, astrocytes, and fibroblast-like cells when treated with RA. Treatment of the cells with dimethyl sulfoxide leads to differentiation along mesodermal lineages, including skeletal and cardiac muscle. We have used the P19 cell line to study the Wnt-1 promoter and identify and characterize the transcription factor(s) that regulates the differentiation-specific transcription of Wnt-1 in RA-treated P19 cultures. Transient-transfection assays have revealed that a 230-bp region comprising positions -278 to -47 of the 5' upstream Wnt-1 sequence was sufficient to direct RA-specific transcription. This promoter fragment was shown to contain a binding site for a nuclear factor that was not detected in undifferentiated P19 stem cells or their dimethyl sulfoxide-treated derivatives but was induced in differentiating RA-treated cells. This factor was termed Wnt-1-inducing factor-1 (WiF-1). DNase I footprinting analysis has identified the G/C-rich WiF-1 binding site, and UV cross-linking studies have shown that WiF-1 is a protein with an M(r) of 65,000. WiF-1 binding activity was also detected in postpubertal mouse testis, the only tissue that expresses Wnt-1 in adults. Site-directed mutations that inhibited WiF-1 binding to the Wnt-1 promoter concomitantly abolished the activity of the promoter in RA-treated P19 cells. The active WiF-1 protein was purified by DNA affinity chromatography. Our data suggest that WiF-1 is a novel G/C box-binding transcription factor and support a physiological role for WiF-1 in the developmentally regulated expression of Wnt-1.

Animals↗
PubMed2005

Identification and characterization of SSTK, a serine/threonine protein kinase essential for male fertility.

Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1beta, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.

Amino Acid Sequence↗
PubMed1988

Developmentally regulated expression of a human "finger"-containing gene encoded by the 5' half of the ret transforming gene.

We isolated and sequenced a cDNA clone of the human gene encoded by the 5' half of the ret transforming gene. The nucleotide sequence indicates that it encodes a protein with "finger" structures which represent putative metal- and nucleic acid-binding domains. Transcription of this gene was detected at high levels in a variety of human and rodent tumor cell lines, mouse testis, and embryos. In addition, a unique transcript was observed in testis RNA. When the expression of the unique transcript was examined at different stages of spermatogenesis, a striking increase in mRNA levels accompanied progression from meiotic prophase pachytene spermatocytes to postmeiotic round spermatids. This finger-containing gene may thus function in male germ cell development.

Amino Acid Sequence↗
PubMed2002

Blockade of in vivo VEGF-mediated angiogenesis by antisense gene therapy: role of Flk-1 and Flt-1 receptors.

Angiogenesis, the formation of new blood vessels from preexisting ones, is a critical component of various pathologies such as tumor progression, rheumatoid arthritis, and retinopathies. Vascular endothelial growth factor (VEGF) is a mitogenic and chimiotactic factor capable of inducing angiogenesis through the activation of its receptors, fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1), expressed on endothelial cells. The purpose of the present study was to assess if a treatment with antisense (AS) oligonucleotides directed against VEGF receptors Flk-1 or Flt-1 mRNA could prevent VEGF-mediated angiogenesis. With the use of miniosmotic pumps, phosphate-buffered saline, VEGF, or VEGF combined with AS-Flk-1, AS-Flt-1, or AS-scrambled oligonucleotides were released in mouse testis for 14 days. VEGF (1, 2.5, and 5 microg) increased the formation of new capillary blood vessels by 236, 246, and 287%, respectively. The combination of AS-Flk-1 or AS-Flt-1 (200 microg) to VEGF (2.5 microg) reduced by 87 and 85% the formation of new blood vessels, respectively, and the expression of their corresponding proteins. These data demonstrate the therapeutical potential of AS-Flk-1 or AS-Flt-1 to prevent VEGF-mediated angiogenesis in vivo.

Animals↗