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PubMed1987

Purification and characterization of poly (ADP-ribose) synthetase from human placenta.

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.

Animals↗
PubMed1989

[Photodynamic effect of hematoporphyrin on cell organs].

Seminiferous epithelial cells of mouse testis were treated with hematoporphyrin derivative (HPD)-light in vitro. After HPD-light treatment, the changes within cells in early stage of prophase were observed. Examination with EM showed that 30 min after HPD-light treatment, the mitochondria were aggregated and vesiculated, the nucleus showed an irregular outline and a sign of chromatolysis; the other organelles showed no significant changes at this time. The results suggest that both mitochondria and nucleus are susceptible to HPD-light treatment. Three hours after HPD-light treatment, many of multi-nucleated spermatids appeared and they all contained deformed nuclei. This fact suggests that cytokinesis was inhibited by this treatment, and the chromatin of metaphase were also damage.

Animals↗
PubMed1989

P150c-abl is detected in mouse male germ cells by an in vitro kinase assay and is associated with stage-specific phosphoproteins in haploid cells.

We have utilized c-abl antibodies and an in vitro kinase assay to identify the protein products of the c-abl gene in mouse testis. Although the testis contains high levels of a unique c-abl mRNA, along with lower amounts of two c-abl mRNAs common to somatic cells, we detected only a single polypeptide of approximately 150 kd, indistinguishable by our methods from P150c-abl observed in murine tissues and cell lines. P150c-abl was also detected in enriched populations of germ cells, including late stage spermatids which contain the highest levels of the novel c-abl transcript. In mature testis, and specifically in late spermatids, P150c-abl co-precipitated with phosphoproteins of approximately 74 kd which were labeled during the in vitro kinase assay. These proteins were phosphorylated predominantly on serine and their phosphopeptide maps differed from that of P150c-abl. The P74 phosphoproteins were not found in association with P150c-abl in germ cells at earlier developmental stages, nor in other tissues or cell lines examined.

Amino Acids↗
PubMed1989

[Intratesticular stimulation of spermatogenesis].

The possibility to exert local effect on suppressed spermatogenesis in patients with different forms of secretory sterility is presented. To this end, the authors created an experimental model of 10-minute acute ischemia in mouse testis. Destructive changes in the testicular parenchyma after the 3., 9., 22. and 30 day were observed and described. Testosterone concentration in the tissues of the ischemic and the contralateral testis was determined by highly effective liquid chromatography. It is concluded that saturation of the interstitium with androgens may avert irreversible postischemic sequelae in the testis. At first chorion gonadotropin and testosterone, but later on Sustanon-250 with prolonged action, were used as protectors. This experimental study is a contribution both to the investigation of spermatogenesis physiology and disturbances and to the refinement of the treatment of secretory forms of male sterility.

Animals↗
PubMed1988

[Calmodulin and neuron: immunohistochemical studies].

Calmodulin is present in higher concentrations in brain tissues. The content rapidly increased during the 2nd postnatal week in rat brain. Although the protein is ubiquitous in all eukaryotic cells, immunohistochemical studies have revealed that calmodulin is mainly localized in the neurons, exhibiting a similar distribution to that of gamma-type neuron-specific enolase. In the mouse retina, both calmodulin and gamma-enolase were found to be localized in optic nerves, ganglion cells, and inner and outer plexiform layers. The development study showed that gamma-enolase increased in the 2nd postnatal week and that the levels of calmodulin did not significantly change in that stage. In the mouse retina with an inherited retinal dysplasia (C3H), in which all the photoreceptor cells degenerate during the 2nd and 3rd postnatal weeks, calmodulin-specific staining decreased in the residual layers. Calmodulin is also enriched in mammalian testes. In the mouse testis, levels of calmodulin were high in the spermatocytes and in the spermatids, as compared to the level in spermatozoa. This suggests that the large amount of calmodulin in the testis may be associated with miotic divisions and/or spermatogenesis. Immunocytochemical staining of calmodulin in C6 glioma cells and PC12 pheochromocytoma cells showed a high level of calmodulin to be localized on the half spindles between poles and chromosomes in mitotic cells. The protein was also shown to be localized on fibrous structures in the interphase of those cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Morphologic analysis of spontaneous teratocarcinogenesis in developing testes of strain 129/Sv-ter mice.

Spontaneous teratocarcinogenesis in the mouse testis begins during the early stages of gonad differentiation. Using inbred strain 129/Sv-ter mice which are highly susceptible to these tumors, the authors have examined the morphologic features of the testis during the gestational period defined from Day 13 through birth. Normal inbred mice (129/J) and random bred mice (Swiss Webster, SW) were used as control groups. Serially sectioned gonads were evaluated at the light- and electron-microscopic levels for histologic changes. In agreement with studies by other workers, embryonal carcinoma cells (ECCs) were observed in tumor-susceptible mice. Cellular arrangements varied from vesicular to nodular. Cell death within advanced tumors was labeled "apoptosis" (shrinkage necrosis). Also encountered were syncytial arrangements of gonocytes (atypical gonocytes), which were present in all animal groups. The significance of atypical gonocytes in relation to degeneration and preneoplasia is addressed.

Animals↗
PubMedDate not supplied

[Relative biological effectiveness of tritium oxide by the criterion of death of the germ cells in mice].

It was shown that low-level beta-radiation of tritium is much more effective than gamma-radiation of 137Cs with respect to reduction of the mouse testis weight. The RBE coefficient increases from 1.8 at a dose of 1 Gy to 2.2-2.3 at 0.1 Gy. On the basis of the data obtained by the authors and those reported in the literature a quality factor is proposed for tritium: QF = 2. Using THO and Na36Cl labels a mean water content of the testis cells, necessary for the estimation of a tritium-radiation dose absorbed, has been determined: gamma ct = 0.70 +/- 0.02 ml/g.

Animals↗
PubMed1984

Characterization of a cell-surface differentiation antigen of mouse spermatogenesis: timing and localization of expression by immunohistochemistry using a monoclonal antibody.

The XT-1 antigen, bound by monoclonal antibody XT-I, is a differentiation antigen of germ cells in the mouse testis. As seen in immunoperoxidase-stained tissue sections from several juvenile ages and adult, the antigen becomes detectable on early (leptotene/zygotene) spermatocytes and increases in staining during spermatocyte development. During spermatid development the distribution of the determinant shifts from its relatively uniform surface distribution on spermatocytes to a more restricted localization on the base of the head, tail and cytoplasmic lobe of the elongating spermatid. The antigen is not detectable on juvenile or adult Sertoli cells. Detection of the antigen is dependent on the presence of germ cells of appropriate developmental stage. It is, thus, a marker for spermatocytes and later germ cells, for a cell-surface molecule related to spermatogenesis and for redistribution and/or modification of the molecule during spermatid elongation.

Age Factors↗
PubMed1993

Construction of subtractive cDNA libraries from limited amounts of mRNA and multiple cycles of subtraction.

A new strategy to construct subtractive cDNA libraries was developed. The method was based on multiple subtraction cycles from small amounts of RNA using paramagnetic technology. An additional application of this technique is the potential to recover (and reuse) the same mRNAs or the single-stranded cDNA from the solid paramagnetic support. The result was assessed in the generation of a specific prepuberal mouse testis library.

Animals↗
PubMed1995

Expression of protein kinase C delta gene in germ cells.

PURPOSE: To investigate the biological functions of protein kinase C delta (PKC delta) in spermatogenesis. MATERIALS AND METHODS: We examined PKC delta transcript in mouse testis by means of in situ hybridization and Northern blotting. RESULTS: In testes of normal mice, signals of PKC delta gene expression were detected specifically at the spermatid stage. The PKC delta gene was weakly expressed in 8-week-old mice and highly expressed by 12 weeks. However, the expression was not detected in testes of germ cell-deficient W/Wv mice even at 12 weeks. CONCLUSIONS: Protein kinase C delta gene expression may be controlled by specific developmental processes and PKC delta may play a role in spermatogenesis.

Animals↗
PubMed1995

Radioprotection against biological effects of internal radionuclides in vivo by S-(2-aminoethyl)isothiouronium bromide hydrobromide (AET).

UNLABELLED: Radionuclides employed in diagnostic and therapeutic nuclear medicine impart radiation energy to tissue over an extended period of time, which depends on the physical half-life and the biological properties of the radiochemical employed. It is therefore important to examine the capacity of chemical radioprotectors to mitigate damage caused by chronic irradiation by incorporated radionuclides. METHODS: Spermatogenesis in mouse testis is used as the experimental model, and spermatogonial cell survival as measured by testicular spermhead count is the biological end point. The capacity of S-(2-aminoethyl)isothiouronium bromide hydrobromide (AET) to mitigate radiation damage caused by chronic irradiation by the radiochemicals 125IUdR, H125IPDM and 210Po-citrate, is investigated. RESULTS: The radioprotection provided by AET is substantial and similar for both of the radioiodinated compounds with dose modification factors (DMF) of 4.0 +/- 1.2 for 125IUdR and 3.4 +/- 0.4 for H125IPDM. In contrast, the damage caused by 210Po alpha particles is protected against to a lesser degree (DMF = 2.4 +/- 0.5). CONCLUSION: The present radioprotection data for AET, in conjunction with our earlier findings for the chemical protectors cysteamine and vitamin C in the same experimental model, suggest that such compounds may be clinically useful as mitigating agents against biological damage caused by incorporated radionuclides. The observed DMFs for AET also support our earlier premise that the mechanism by which DNA-incorporated Auger emitters impart biological damage is primarily radical mediated, and hence indirect in nature.

Animals↗
PubMed1994

Molecular cloning of a novel Ca(2+)-binding protein (calmegin) specifically expressed during male meiotic germ cell development.

During mammalian spermatogenesis, many specific molecules are expressed. We have recently identified a 93-kDa male meiotic germ cell-specific antigen (Meg 1) exclusively expressed in germ cells from the pachytene spermatocyte to the spermatid stage using the monoclonal antibody TRA 369 (Watanabe, D., Sawada, K., Koshimizu, U., Kagawa, T., and Nishimune, Y. (1992) Mol. Reprod. Dev. 33, 307-312). In this study, we cloned a cDNA representing this antigen from a mouse testis cDNA expression library, using the monoclonal antibody TRA 369. Northern blotting showed that this transcript was 2.3 kilobases in length and was expressed only in the testis and not in other somatic tissues or in the ovary. The expression of the mRNA was first detected at the pachytene spermatocyte stage of male germ cell development, and this expression was correlated with the expression of the protein. Sequence analysis of the cDNA revealed that the predicted protein consists of 611 amino acids, including a hydrophobic NH2 terminus characteristic of a signal peptide, two sets of internal repetitive sequences (four repeats of IPDPSAVKPEDWDD and GEWXPPMIPNPXYQ), and a hydrophilic COOH terminus. The deduced amino acid sequence has 58% homology with dog calnexin (the ER membrane phosphoprotein of pancreatic cells) and significant partial homology with calreticulin (high affinity Ca(2+)-binding protein of the ER membrane) at the repetitive sequence. Furthermore, we demonstrated the 45Ca2+ binding ability of this antigen by a 45Ca2+ overlay assay, and the name calmegin is proposed for this antigen. Calmegin is a novel Ca(2+)-binding protein that is specifically expressed in spermatogenesis. The highly regulated, specific, and abundant expression of calmegin suggests that it has important roles in spermatogenesis.

Amino Acid Sequence↗
PubMedDate not supplied

[An ultrastructural analysis of the testes in mice subjected to long-term exposure to a 17-kHz electrical field].

The effect of long-term (9 months) exposure to electric field (17 kHz, 100 kV/m) on mouse testis has been investigated. The increase in destructive processes was noted in both spermatogenous epithelium cells and in steroid-producing cells; the histohematic barrier status changed. The results obtained indicate premature reproductive ageing of animals under the effect of electric field.

Animals↗
PubMed1993

On the equivalent dose for Auger electron emitters.

Radionuclides that emit Auger electrons are widely used in nuclear medicine (e.g., 99mTc, 123I, 201Tl) and biomedical research (e.g., 51Cr, 125I), and they are present in the environment (e.g., 40K, 55Fe). Depending on the subcellular distribution of the radionuclide, the biological effects caused by tissue-incorporated Auger emitters can be as severe as those from high-LET alpha particles. However, the recently adopted recommendations of the International Commission on Radiological Protection (ICRP) provide no guidance with regard to calculating the equivalent dose for these radionuclides. The present work, using spermatogenesis in mouse testis as the experimental model, shows that the lethality of the prolific Auger emitter 125I is linearly dependent on the fraction of the radioactivity in the organ that is bound to DNA. This suggests that the equivalent dose for Auger emitters may have a similar linear dependence. Accordingly, a formalism for calculating the equivalent dose for Auger emitters is advanced within the ICRP framework.

Animals↗
PubMed1996

Exon circularization in mammalian nuclear extracts.

Correct ligation of exons in pre-mRNA splicing requires splice site juxtaposition (splice site pairing), usually involving a 5' splice site and a downstream 3' splice site. Splicing of a 5' splice site to an upstream 3' splice site, however, is predicted to result in a circular RNA. This mode of splice site pairing across the axon has been hypothesized to account for rare RNAs containing scrambled exons (Nigro JM et al., 1991, Celt 64:607-613; Cocquerelle C et al., 1992, EMBO J 11:1 095-1098). Additionally, this mode of splice site pairing has been postulated to explain the formation of SRY circular transcripts in mouse testis (Capel B et al., 1993, Celt 73:1019- 1030). Here we show that splice site pairing across the exon can result in exon circularization in vitro. These results indicate that spliceosome-mediated axon circularization indeed can account for the formation of scrambled exons and circular RNAs. Exon circularization efficiency decreased dramatically as the length of the exon was increased from 95 nt to 274 nt. Circularization of this longer exon was restored, however, when intronic complementary sequences were included in the RNA substrate. These complementary sequences could form a stem that served to bring the splice sites into proximity and thereby promote splice site pairing. Therefore, the splicing of this structured RNA recapitulated SRY-like exon circularization in vitro.

Base Sequence↗
PubMed1995

The lactate dehydrogenase gene from nematode Caenorhabditis elegans contains only two of six introns conserved in the protein-encoding sequence of LDH genes from bird and mammals.

The protein-encoding region of L-lactate dehydrogenase (LDH) gene from nematode, Caenorhabditis elegans, was amplified by polymerase-chain-reaction from total genomic DNA and its nucleotide sequence determined. A comparison of this genomic sequence with the published sequence of nematode LDH cDNA reveals the presence of two introns of 57 and 47 nucleotides at codon no. 82 and 279-280, respectively. The positions of the two introns present in this invertebrate LDH gene correspond to the second and sixth introns of vertebrate LDH genes. The protein-coding sequence of human LDH-A (muscle), LDH-B (heart) and LDH-C (testis), mouse LDH-A, and duck LDH-B genes has previously been shown to be interrupted by six introns at the homologous positions.

Amino Acid Sequence↗
PubMedDate not supplied

Developmental expression of glutathione peroxidase, catalase, and manganese superoxide dismutase mRNAs during spermatogenesis in the mouse.

We have examined in mouse testis the steady-state levels of mRNAs encoding glutathione peroxidase (GSHPx), catalase (CAT), and superoxide dismutase 2 (SOD-2), three enzymes essential for the antioxidant protection of cells. In RNA preparations derived from prepuberal and adult testes and from isolated populations of meiotic and post-meiotic germ cells, one major GSHPx mRNA of about 0.8 kilobases (kb) and one major CAT mRNA of about 2.4 kb were detected. Three SOD-2 mRNAs of about 2.2, 1.2, and 1.0 kb were found in testis. In contrast to GSHPx and CAT, the mRNA levels of SOD-2 were higher in testis than in liver. SOD-2 mRNA levels are developmentally and translationally regulated with maximal levels of expression in early post-meiotic germ cells, whereas the levels of GSHPx and CAT mRNAs are relatively constant in both prepuberal and adult testes. These data suggest that translational regulation plays a more prominent role for SOD-2 expression than for GSHPx or CAT expression in the mammalian testis.

Animals↗
PubMedDate not supplied

P450 aromatase messenger ribonucleic acid expression in male rat germ cells: detection by reverse transcription-polymerase chain reaction amplification.

We have previously demonstrated that cytochrome P450 aromatase (P450arom) protein, an estrogen-synthesizing enzyme, is present and active in germ cells of the adult mouse testis. To establish that P450arom mRNA is expressed in germ cells of other species, we examined expression of P450arom in adult rat germ cells by employing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from Staput separated germ cells and reverse transcribed. The resulting cDNA was amplified by nested PCR reactions using oligonucleotide primers selected from a highly conserved region of the P450arom gene. RT-PCR analysis yielded cDNA products of 334 bp in length that corresponded to the predicted size expected from the final nested amplification. The identity of the germ cell P450arom PCR products was confirmed by restriction enzyme analysis and direct nucleotide sequencing. Rat genomic DNA was subjected to PCR to verify that P450arom DNA products were not obtained from genomic DNA contamination. Rat genomic DNA yielded a nested PCR product for P450arom of approximately 2000 bp, suggesting that, as is the case with the human P450arom gene, the rat P450arom gene contains an intron in the amplified region. In addition, a semiquantitative technique was utilized to eliminate the possibility that the P450arom RT-PCR products were derived from Leydig cell contamination of Sta-put-separated germ cell preparations. RT-PCR for P450arom and 3-beta-hydroxysteroid dehydrogenase (3 beta-HSD), a Leydig cell-specific steroidogenic enzyme, was carried out on Sta-put-separated germ cells and interstitial cell preparations containing Leydig cells. P450arom and 3 beta-HSD RT-PCR reactions were stopped at three cycle intervals to detect and compare the earliest appearance of RT-PCR reaction products in various cell types. Results indicated that P450arom mRNA is detected in round spermatids before it is detected in interstitial cells, whereas 3 beta-HSD was detected only in interstitial cells, suggesting that the P450arom mRNA detected in germ cells is not due to interstitial cell contamination of germ cell preparations. Therefore, our results indicate that P450arom mRNA is expressed in adult rat germ cells and that testicular germ cells are a potential source of estrogen in the male reproductive tract.

Amino Acid Sequence↗