Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Normal development of hamster and rabbit eggs fertilized by spermatozoa labelled with the fluorescent thiol alkylating agent, monobromobimane

Cauda epididymal spermatozoa of the golden hamster were labelled with the thiol-alkylating reagent, monobromobimane (MB). Female hamsters underwent uterine insemination with labelled spermatozoa at laparotomy under metofane anesthesia. All 12 females examined between 5 and 54 h postinsemination yielded a total of 83/100 (83%) eggs in the process of fertilization or embryos. Under ultraviolet (UV) exposure all exhibited a fluorescent tail which, in the 4- and 8-cell embryos, could be seen to be fraying or disintegrating. As cleavage progressed, labelled tail components came to be restricted among the blastomeres such that at the 4- and 8-cell stage the tail could be seen in only one to three blastomeres. To study complete development and pregnancy another 12 females received uterine insemination. After recovery from anesthesia (approximately equal to 4 h) these females were mated with a vasectomized male bearing a dominant genetic marker (black eyes) to allow unequivocal determination of paternity in the fetuses and young produced. Seven became pregnant with one female losing her pregnancy about Day 7 of gestation. Two females sacrificed on Day 13 produced 17 normal fetuses and one resorption. Four females delivered 16 young, all normal at birth and in subsequent growth and fertility. In addition, insemination of female rabbits with MB-labelled spermatozoa yielded normal embryos (50/52 96%) from 3 does on Day 2 and (38/64 60%) from 4 does on Days 4 or 5. Two normal litters (9 bunnies) have delivered from 3 does allowed to carry to term.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

We examined the action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G(1) cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate.     

Effects of exposure to sublethal levels of cadmium upon water-electrolyte status in the goldfish (Carassius auratus)

Goldfish were exposed to sublethal levels of cadmium (means of 44.5 and 380 μg Cd⁺⁺/l) for periods of 25 and 50 days, and their water-electrolyte status evaluated by reference to plasma and muscle levels of sodium, potassium and chloride and muscle water content. Significant changes in plasma chloride, tissue potassium and tissue water content were observed after 25 days in both test solutions. Specimens held at the more dilute cadmium concentration were apparently able to compensate for most of the initial cadmium effect and, after 50 days exposure, were characterized only by a continuing depression in plasma sodium level. This suggests that the cadmium MATC value for this species under the conditions employed is probably less than 45 μg Cd⁺⁺/l. Goldfish exposed to 380 μg Cd⁺⁺/l for 50 days exhibited significant deviations in plasma sodium and chloride levels as well as in tissue sodium and water content, and these parameters may provide useful indices of cadmium effects at sublethal concentrations.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations. II. Photoreactivation and killing of rat kangaroo cells (Potorous tridactylus) and Herpes simplex virus-1 by exposure to fluorescent "white" light or sunlight

Photoreactivation (PR) of ultraviolet (254 nm)-inactivated cornea cells of the potoroo (or rat kangaroo; Potorous tridacylus) has been studied at wavelengths greater than 375 nm from either fluorescent "white" light or sunlight. In both cases the PR kinetics curves pass through maxima, which most likely result from the superposition of concomitant inactivation by the photoreactivating light. The inactivating effect of light was directly demonstrated for non-UV-irradiated cells, permitting correction of the PR curves. Wavelengths greater than 475 nm, and even greater than 560 nm, which do not noticeably damage cells, still photoreactivate, though less effectively than shorter wavelengths. Light treatment of UV-inactivated Herpes simplex Virus-1 (HSV-1) after infection leads to PR effects resembling those observed for cells, while light treatment of unirradiated virus after infection likewise causes inactivation. The "fluence-reduction factor" of PR, which is greater than 3 for the virus, exceeds that for the cells, where it decreases with increasing UV fluence. In vitro tests have indicated that sunlight greater than 375 nm causes photorepairable DNA lesions which are virtually fully repaired by the same light. Thus cell inactivation resulting from these solar wavelengths must be due to non-photorepairable damage.     

Chromosomal analysis in mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) and methyl and ethyl methanesulfonate (MMS and EMS)

Chromosome aberrations were analyzed at the first-cleavage metaphase of mouse eggs fertilized in vitro with sperm exposed to ultraviolet light (UV) as well as to methyl and ethyl methanesulfonate (MMS and EMS). The frequencies of chromosome aberrations markedly increased with dose of UV as well as with concentration of MMS and EMS. In the UV-irradiation group, the frequency of chromosome-type aberrations was much higher than that of chromatid-type aberrations. About 90% of chromosome aberrations observed in the eggs following MMS and EMS treatment to sperm were chromosome type in which the frequency of chromosome fragments was the highest. The effects of UV on the induction of chromosome aberrations were clearly potentiated by post-treatment incubation of fertilized eggs in the presence of Ara-C or caffeine, but the effects of MMS and EMS were not pronounced by post-treatment of Ara-C or caffeine. The results indicate a possibility that UV damage induced in mouse sperm DNA is reparable in the eggs during the period between the entry of sperm into the egg cytoplasm and the first-cleavage metaphase.     

Trophospongium-like structures in an insect eye: Response of retinula cells of Papilio xuthus (Lepidoptera) to irradiation with ultraviolet light

Cell and Tissue Research - Presumably, as a consequence of a 3-h exposure to light of 350-nm wavelength (1.5 × 1015 photons/cm2 sec) followed by a period of 20 h of rest in the dark, the four...     

"Intermediate zone" of mammalian spleens: light and electron microscopic study of three primitive mammalian species (platypus, shrew, and mole) with special reference to intrasplenic arteriovenous communication

The intermediate zone (IZ) of nonperfused and perfused spleens in three species of primitive mammals (shrew, mole, platypus) was studied morphologically. The IZ is a tissue zone consisting of plexiform vessels, probably venous capillaries, and is located transitionally between the white and red pulp. The IZ is separated from the white pulp by the arterial net (AN), in which the white pulp arteries terminate. Development of the IZ differs between the three species examined being distinctive in the platypus and shrew. The IZ is thin in the mole spleen. A closed type of arteriovenous (A-V) anastomosis was demonstrated in or around the IZ in the two Insectivora species examined. In the shrew spleen, peripheral arterial branches running within the IZ anastomose with the AN around the follicle. The AN anastomoses eventually with venous plexiform vessels of the IZ around the nonfollicular area of the white pulp to form a closed system. In the mole spleen, A-V anastomoses were noted between white pulp arteries (follicular and AN) and veins of the red pulp, either by direct communication or through fenestrated IZ vessels compatible with the plexiform vessels of the shrew spleen. A-V anastomosis in the IZ is probable, but not confirmed, in the platypus spleen, as analysis was limited to a nonperfused specimen. Well-developed ellipsoids were noted around arterial terminals of the IZ in the shrew spleen. Ellipsoids were also noted around all arterial terminals of the mole spleen directed to the red pulp. Most ellipsoids of the mole spleen appeared located within the IZ. No ellipsoids were present around arterial terminals of the IZ in the platypus spleen. Closed circulation was noted in terminals of the pulp artery in spleens of all three species. All pulp arteries of the mole spleen are postellipsoid segments of white pulp (AN and follicle) arteries. No ellipsoids were found around terminals of the pulp artery (penicillar artery) in shrew and platypus spleens. The IZ is probably homologous to the perilymphatic sinusoid (vein) of the lungfish spleen and may be regarded as part of the red pulp. The IZ may be representative of primitive mammalian spleens that have closed circulation. The marginal zone (MZ) of common mammalian spleens is probably a modified IZ by differentiation (remodelling) of the intrasplenic vein. In this process, withdrawal of venous vessels from the IZ occurred, leaving a lymphoreticular zone with open circulation (MZ). The marginal sinus reported in some mammalian spleens is probably a modified AN formed during this process. Possible morphological alterations of the spleen in vertebrate phylogeny are discussed.     

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.     

Chromosome analysis of human spermatozoa following in vitro exposure to cyclophosphamide, benzo(a)pyrene and N-nitrosodimethylamine in the presence of rat liver S9

For the purpose of assessing mutagenic effects (clastogenicity) of metabolites derived from chemical mutagens/carcinogens on human sperm chromosomes, spermatozoa were exposed in vitro to cyclophosphamide (CP), benzo(a)pyrene (BP) or N-nitrosodimethylamine (NDMA) for 2h in the presence or absence of rat liver S9, a metabolic activator of these chemicals. After in vitro fertilization between human spermatozoa and zona-free hamster oocytes, chromosome complements of sperm origin were analyzed cytogenetically.In the absence of S9, none of three chemicals (20 microg/ml CP, 200 microg/ml BP and 20mg/ml NDMA) caused a significant increase in spermatozoa with structural chromosome aberrations (8.6, 10.0 and 7.5%), as compared with their matched controls (10.9, 11.0 and 8.5%). In the presence of S9, however, a significant increase in chromosomally abnormal spermatozoa was observed in CP (37.1%, P < 0.001) and BP (31.0%, P < 0.001), indicating that enzymatic activation of CP and BP induced chromosomal abnormalities in human sperm. In contrast, NDMA did not induce chromosome aberrations in human spermatozoa by S9 treatment, although positive results have been observed in somatic cells. The present results on in vitro clastogenicity of CP, BP and NDMA are consistent with the results in previous in vivo studies with murine spermatozoa. Our S9/human sperm chromosome assay seems to be useful for estimation of hereditary risk of chemicals in human. Because most chemicals need metabolic activation to bind to DNA.     

Interaction of insulin analogs, glucagon, growth hormone, vasopressin, oxytocin, and scrambled forms of ribonuclease and lysozyme with glytathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase): dependence upon conformation.

Interactions of several proteins with glutathione-insulin transhydrogenase (GIT) have been investigated by determining their ability to inhibit degradation of 125I-labeled insulin catalyzed by GIT. The inhibition by every insulin analog (des-Asn-des-Ala-pork insulin, desoctapeptide-pork insulin, des-Ala-pork insulin, pork insulin, proinsulin, and guinea pig insulin) was competitive vs. competitive vs. insulin indicating that they function as alternate substrates. The insulin analogs with the least hormonal activity showed the highest potency as inhigitors of insulin degradation. Whereas native ribonuclease and lysozyme showed little or no inhibition, their scrambled forms (i.e. reduced and randomly reoxidized) showed competitive inhibition with a potency greater than that of insulin. These results suggest that the conformation of the substrate or inhibitor is probably the major factor in determining the specificity for (or binding to) the enzyme. Studies withother peptide hormones showed competitive inhibition with vasopressin and oxytocin and noncompetitive inhibition with glycagon. The inhibition with growth hormone could be either competitive or noncompetitive. The inhibition by glucagon and growth hormone (physiologic antagonists of insulin) could serve as a control mechanism to modulate the activity of enzyme. The following showed very little or no inhibition; the native and scrambled form of pepsinogen, trypsin inhibitor of beef pancreas and of lima bean, C-peptide of pork proinsulin, and heptapeptide (B23-B29) of insulin.     

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae,Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid or nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.     

Chromosome analysis of mouse zygotes after injecting oocytes with spermatozoa treated in vitro with green tea catechin, (-)-epigallocatechin gallate (EGCG)

The cytogenetic effects of (-)-epigallocatechin gallate (EGCG) on mouse spermatozoa were studied in vitro using an intracytoplasmic sperm injection (ICSI) technique. Spermatozoa were collected by the swim-up method and treated with EGCG at 1 microM and 10 microM. When motile, EGCG-treated spermatozoa were injected into oocytes, structural chromosome aberrations (SCAs) at the first cleavage metaphase did not increase significantly. However, a majority of immotile spermatozoa treated with 10 microM EGCG had the following abnormalities: pronuclear arrest (11% of activated oocytes), degenerated sperm chromatin (chromosome) mass (30% of activated oocytes) and occurrence of structural chromosome aberrations (57% of analyzed metaphases). The incidence of these abnormalities suggests that immotile spermatozoa were susceptible to EGCG, and that the damage of sperm chromatin was accelerated in immotile spermatozoa by 10 microM EGCG treatment.     

Effects of Long-Term exposure to Heavy Metals upon Rhizosphere Bacteria from Baia Mare Area (Maramureş County,Romania)

Abstract

The aim of this study was to investigate the extent of heavy metal (HM) pollution and its effect on microorganisms from rhizosphere soil in Baia Mare area (Maramure? County, Romania). Two sites with different contamination degrees were included in the study: one with a long history of mining activities and one within a drinking water safeguard zone. Rhizosphere soil samples were characterized with respect to physico-chemical parameters and the Cd, Cu, Pb and Zn contents. Native bacteria were investigated for HM tolerance and biofilm formation under toxic exposure by the microdilution assay. The most resistant strains were identified and the minimum inhibitory concentrations for HMs were determined. Cd, Cu, Pb and Zn exceeded the intervention threshold in Bozânta tailings site, while Pb content exceeded the intervention level within the area of the drinking water treatment plant. Cd showed a very high potential ecological risk in Bozânta area. The long-term exposure to HMs contributed to the selection of HM-tolerant and weakly adherent strains. Biofouling was significantly reduced under the influence of copper ions. Arthrobacter, Rhodococcus and Acidovorax strains with exceptional resistant profiles were isolated from the tailings site, indicating the important role of native microorganisms in rhizosphere ecosystems of contaminated sites.     

Karyotypic characterization of representatives from Melolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fluorescent in situ hybridization (FISH)

Meiotic chromosomes of Phyllophaga (Phytalus) vestita, Phyllophaga (Phyllophaga) aff capillata and Lyogenys fuscus (Melolonthinae) were analyzed by conventional staining, C-banding, fluorochromes, silver nitrate and FISH. The three species had a diploid number of 2n=20 and a sex mechanism of the (Xyp; XYp) parachute type. P. (Phytalus) vestita,P. (Phyllophaga) aff capillata and Lyogenys fuscus showed pericentromeric constitutive heterochromatin (CH) in all autosomal bivalents and on X chromosomes. Staining with CMA3 and DAPI fluorochromes showed that the CH of P. (Phytalus) vestita is not specifically rich in AT and GC-base pairs, whereas in P. (Phyllophaga) aff capillata the sex bivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys fuscus CH was positive for DAPI. Silver nitrate staining revealed nucleolar remnants in all three species. However, FISH obtained a precise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization was seen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a small autosomal bivalent of P. (Phyllophaga) aff capillata.     

Partitioning of leaf nitrogen with respect to within canopy light exposure and nitrogen availability in peach (Prunus persica)

Summary Relationships between leaf nitrogen content and within canopy light exposure were studied in mature nectarine peach trees (Prunus persica cv. Fantasia) that had received 0, 112, 196, 280 or 364 kg of fertilizer nitrogen per hectare per year for the previous 3 years. The relationships between light saturated leaf CO₂ assimilation rates and leaf nitrogen concentration were also determined on trees in the highest and lowest nitrogen fertilization treatments. The slope of the linear relationship between leaf N content per unit leaf area and light exposure was similar for all nitrogen treatments but the y-intercept of the relationship increased with increasing N status. The slope of the relationship between leaf N content per unit leaf area and light saturated CO₂ assimilation rates was greater for the high N trees than the low N trees, but maximum measured leaf CO₂ assimilation rates were similar for both the high and low N treatments. A diagrammatic model of the partitioning of leaf photosynthetic capacity with respect to leaf light exposure for high and low nitrogen trees suggests that the major influence of increased N availability is an increase in the photosynthetic capacity of partially shaded leaves but not of the maximum capacity of highly exposed leaves.