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H V Malling

Publications and source records attributed to H V Malling.

At least 37 records · Page 2Linked to original sources
PubMed1989

Differentiation of binuclear spermatozoa in mice.

In an electron microscopy study of abnormal spermatogenesis in mice, we have found that two discrete haploid nuclei may be located in a single spermatid cytoplasm after the second meiotic division. The spermatid continues to differentiate and forms a binucleate spermatozoon with both nuclei separately packaged within the sperm head. The Golgi apparatus of the double spermatid forms a single proacrosome that attaches to both nuclei. Apparently, one acrosomal structure differentiates to cover and compartmentalize the two haploid nuclei within the sperm head. Chromatin condensation appears normal. The head morphology and number of flagella vary in mature spermatozoa produced by this process. This work demonstrates one pathway by which polyploid spermatids continue to differentiate to spermatozoa after failure of cytoplasmic division or possibly cellular fusion.

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PubMed1989

Use of phi X174 as a shuttle vector for the study of in vivo mammalian mutagenesis.

The most promising new techniques for the study of in vivo mammalian mutagenesis make use of transgenic mice carrying a recoverable vector. Mutation systems in mammals can be based on the selection of altered phenotypes among cells sampled from the whole animal, but they are then limited to the very few cell types in which the marker gene is expressed. Such systems require both in vivo and in vitro cell proliferation for expression and verification of the mutations. To avoid these complications, the study of mutations in most tissues must be based on the detection of genetic alterations in a vector that is independent of the phenotype of the mammalian cell. The vector is only a small portion of the mammalian genome, and many of the procedures for recovering the vector are inhibited by the host DNA. For this reason, partial purification is necessary. The purification is made possible by using vectors which are not cut by restriction enzymes that cut the host DNA to pieces of an average size considerably smaller than the vector. The efficiency for measuring mutation frequencies depends on the number of vectors which can be recovered from a certain amount of DNA and is affected by the number of vectors per mammalian genome and the transfection efficiency of the partially-purified vector. In order to avoid selection against or for the spontaneous or induced mutations, the transfection efficiency of the vector from the transformed DNA and of the pure vector DNA should be of the same order of magnitude. Differences in the response to mutagens between the mammalian genome and the procaryotic vector may be expected due to the lack of unique mammalian topographical features in the vectors. Any mutation induction which depends preferentially on these unique features of the mammalian genome may not be detected in a shuttle vector system unless the vector has been engineered or specifically designed to include such topographical characters. The shortcoming of short-term tests that use mutagenicity for predicting human carcinogenicity is usually lack of correlation between mutagenesis in the short-term tests and the corresponding results in carcinogenesis bioassays in mammals. One factor which could contribute to the lack of correlation between the short-term test systems and the bioassays is that we are comparing mutations in totally different genes in different organisms. By using the phi X174 shuttle system, one of the variables may be eliminated.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1989

Mutagenesis of phi X174 am3 cs70 incorporated into the genome of mouse L-cells.

The objective of our work with phi X174 has been to develop a shuttle vector that can be used comparatively in bacterial cells, different types of mammalian cells, and possibly in the various tissues of transgenic mice, with a constant mechanism for detection and analysis of mutations independent of any host-cell type. Toward that end, we have efficiently rescued phi X174 am3 cs70 that is host-silent and stably integrated into the genome of mouse L-cells. The particular mouse L-cell line contains tandem arrays, single copies, and fragments of phi X that, upon restriction enzyme excision, can result in 5 potentially active copies per diploid genome. The excised phi X DNA is recovered by column chromatography, ligated, and transfected into highly competent spheroplasts. The Rescue Efficiency, defined as the number of viable phages produced out of the total number of potentially recoverable copies, is approx. 10(-3). The Recovery Ratio, defined as the Rescue Efficiency for chromosomally-integrated phage DNA divided by the Rescue Efficiency for phi X am3 cs70, is close to one. Mouse L-cells containing the integrated phi X174 am3 cs70 were treated with 20 mM ethyl methanesulfonate. The reversion frequency of am3 among progeny phages rescued from treated cells was 1.4 X 10(-5) (193 revertants in 1.4 X 10(7) phages). This is significantly higher than the 5.8 X 10(-7) reversion frequency of am3 (7 revertants in 1.2 X 10(7) phages) among progeny phages rescued from untreated cells.

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PubMed1983

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts, (6 agents), motility (5 agents), and F1 sperm morphology (7 agents)). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), and (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity approximately equal to 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.

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PubMed1983

An evaluation of human sperm as indicators of chemically induced alterations of spermatogenic function. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

To evaluate the utility of sperm tests as indicators of chemical effects on human spermatogenesis, the literature on 4 sperm tests used to assess chemically induced testicular dysfunction was reviewed. The tests surveyed included sperm count, motility, morphology (seminal cytology), and double Y-body (a fluorescence-based test thought to detect Y-chromosomal nondisjunction). There were 132 papers that provided sufficient data for evaluation. These reports encompassed 89 different chemical exposures: 53 were to single agents; 14 to complex mixtures; and 22 to combinations of 2 or more identified agents. Approximately 85% of the exposures were to experimental or therapeutic drugs, 10% were to occupational or environmental agents, and 5% were to drugs for personal use. The most common sperm parameter studied was sperm count (for 87 of the 89 exposures reviewed). Sperm motility was evaluated for 59 exposures, morphology for 44, and double Y-bodies for only 4. The 89 exposures reviewed were grouped into 4 classes: those which adversely effected spermatogenesis, as measured by one or more of the sperm tests (52); those suggestive of improving semen quality (11); those showing inconclusive evidence of adverse effects from exposure (14); and those showing no significant changes (12). Since the reviewed reports had a large variety of study designs, and since every attempt was made to include all reports with interpretable data, these classifications were based on reviewing committee decisions rather than on uniform statistical criteria. This review gives strong evidence that human sperm tests can be used to identify chemicals that affect sperm production, but because of our limited understanding of underlying mechanisms, the extent to which they can detect mutagens, carcinogens or agents that affect fertility remains uncertain. For the very few agents studied with both human and mouse sperm tests, similar test-responses were seen; thus sperm tests in mice and other laboratory mammals may have a potential role in hazard identification. An overall comparison of the 4 human sperm tests suggests that no one test is biologically more responsive than another; all of them may thus be needed when testing for chemically induced changes from agents of unknown activity. This review also gives evidence that sperm tests can be used to assess the extent and the potential reversibility of induced spermatogenic damage. The reviewing committee recommends further studies to determine (a) the dose-response characteristics of the human sperm tests, (b) details of the reversibility of induced changes with time after exposure, (c) the relative responses in the 4 sperm tests in exposed individuals, (d) the mechanism of action, (e) the biological and genetic implications of chemically induced effects, and (f) the comparison of responses among different species for risk assessment. The reviewing committee outlines specific considerations for planning new sperm studies on chemically exposed men.

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PubMed1982

A hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin.

A simple hemolytic plaque assay for the detection of red blood cells carrying abnormal or mutant hemoglobin is described. The assay is based on the use, as indicator cells, of sheep red blood cells coated with monospecific antibody against the hemoglobin variant, another complement fixing multispecific anti-hemoglobin antibody as developing serum and an antibody against red cell ghosts which can lyse only the red cells of the species being studied and not the indicator sheep red cells. The test red cells are mixed with the indicator sheep red cells, the developing antiserum and the anti-ghost antibody. The mixture is transferred into thin chambers prepared with 2 microscope slides and incubated at room temperature (25 degrees C). The indicator sheep red cells around each test red cell carrying abnormal or mutant hemoglobin are lysed resulting in the formation of a plaque. Instead of anti-hemoglobin-coated sheep red blood cells, protein A-coated sheep red blood cells may be used as indicator cells provided the developing antiserum is monospecific for the hemoglobin variant. When sheep red blood cells coated with an antibody specific for DBA/2J mouse hemoglobin were used in the assay, only red cells from DBA/2J mice (carrying d hemoglobin) formed plaques while C57BL/6J red cells (carrying s hemoglobin) did not.

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PubMed1982

Effect of procarbazine treatment of mice on alpha-glycerolphosphate dehydrogenase activity and frequency of selected abnormalities in sperm.

The in situ activity of mitochondrial alpha-glycerol phosphate dehydrogenase (alpha-GPD) as well as 2 specific sperm abnormalities [headless and disorganization of the mitochondria assembly (PL-type)] have been studied in sperm from mice treated with single doses of procarbazine (100, 200, 400, 600 and 800 mg/kg) at intervals up to 55 days after treatment. The frequency of sperm without active alpha-GPD and the frequency of the morphology variants increased with increasing dose of procarbazine. The variance in the level of alpha-GPD among sperm with active enzyme and the variance between animals also increased with increasing dose. The lowest effective dose was 200 mg/kg and there were no observable effects 85 days after treatment.

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PubMed1981

Development of in vivo somatic mutation system using antibody against hemoglobin: preparation and use of an anti-hemoglobin antibody for identifying C57BL/6 red cells in artificial mixture of DBA/2 and C57BL/6 red cells.

A cellular specific-locus mutation test is described for detecting mutant cells in mammals. The test is based upon the use of specific anti-C57BL/6J mouse hemoglobin antibody that binds hemoglobin "single" (hemoglobin s, present in C57BL/6J mouse) and not hemoglobin "diffuse" (hemoglobin d, present in DBA/2J mouse). Attempts to purify such antibody from pony and rabbit anti-sera through cross-absorption were unsuccessful. Immunization of LP/J mouse with C57BL/6J hemoglobin produced antiserum that reacted with s hemoglobin but not with a d hemoglobin. In a fluorescent antibody technique, this antibody was found to label fixed red blood cells from C57BL/6J mice but not from DBA/2J mice. In a mixture of C57BL/6J and DBA/2J red cells, the C57BL/6J cells could be differentiated by their bright fluorescence from the non-fluorescent DBA/2J cells. Reconstruction experiment with artificial mixtures of DBA/2J and C57BL/6J cells showed that s hemoglobin bearing cells could be detected in DBA/2J red cells at frequencies as small as 0.4 X 10(-6). Thus, the system is sensitive enough to detect d leads to s mutation in DBA/2J mice. Amino acid comparison of the globin chains of s and d hemoglobins shows that our antibody can probably detect mutations leading to a substitution of serine or proline by alanine at beta 20 position and/or a substitution of threonine by alanine at beta 139 position.

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PubMed1980

In vivo germinal mutation detection with "monospecific" antibody against lactate dehydrogenase-X.

This report describes developments toward a cell specific-locus test for measuring point mutations directly in sperm based upon the use of a monospecific antibody against sperm-specific lactate dehydrogenase-X (LDH-X). The antibody recognizes amino acid differences between mouse and rat LDH-X molecules. In general, mouse sperm do not bind the monospecific antibody against rat LDH-X, but a few exceptional mouse sperm do. Such mouse sperm are believed to contain LDH-X molecules in which an amino acid normally present in mouse LDH-X has been exchanged with one present in rat LDH-X at a place where rat and mouse enzymes are antigenically different. Thus, in the fluorescent antibody technique, mouse sperm carrying normal LDH-X do not stain but the sperm containing mutated enzyme do stain. By using this technique, the spontaneous presumptive mutation frequency in DBA/2 mice was found to be 0.43 X 10(-6); it increased to 7.6 X 10(-6) upon procarbazine treatment of the mice.

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PubMed1979

Action of N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide hydrochloride (procarbazine hydrochloride) in the germ tissue of mice: dominant lethal effects.

A study was carried out on the effects of N-isopropyl-alpha-(2-methylhydrazino)-p-toluamide (procarbazine, Natulan) in the dominant lethal test in the mouse. Male mice were dosed once and mated with fresh virgin females each week. The utilization of sperm, treated as spermatids or testicular sperm with 100-800 mg/kg, resulted in significant post- and pre-implantation death of embryos. Fertility was markedly reduced after the injection of 200 mg/kg of procarbazine and over. This is probably due to a cell killing effect, the most sensitive stages being differentiating spermatogonia, type A sermatogonia and resting primary spermatocytes. Total sterility was induced for several weeks with doses of 600 and 800 mg/kg. Up to 12 weeks after treatment the number of females with implants was still significantly lower than controls indicating a severe depletion of spermatogonial cells. The spectrum of effects correlates well with the drug's effect on nucleic acid and protein synthesis.

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PubMed1979

Influence of subcellular fractions of mammalian testes on the mutagenic activity of nitrofurans toward Escherichia coli; presence of a co-mutagen-like factor.

The activation of nitrofurans to mutagenic intermediates by testicular tissue was investigated. AF-2 and nitrofurazone were tested in a microsomal suspension assay with strain E. coli K-12 343/113 as indicator and subcellular fractions from rabbit testes. Different mutation patterns were observed in the presence or absence of testicular homogenate, indicating the presence of different mutagenic intermediates. The frequency of arg+ reversion increased proportionally to the homogenate concentration suggesting that the nitrofurans were activated by testicular components to intermediates that induced base-pair substitutions. Other experiments showed that a component of low molecular weight, present in the soluble fraction of homogenates of testes from rabbits, rats and monkeys, was responsible for the increased mutation frequency. It is concluded that this "co-mutagen-like" factor either alters the metabolism of nitrofurans in E. coli and/or promotes the formation of base-pair substitution-type mutations. This direct interaction between a nonenzymic component of mammalian testes and the mutation induction/expression process in E. coli suggests the role of co-mutagen-like factors in the sensitivity of testes to nitrofurans.

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PubMed1979

Rhesus monkey (Macaca mulatta) model in genetic toxicology mitomycin C clastogenicity in germ cells.

The value of rhesus monkeys (Macaca mulatta) as a genetic toxicology model is limited by their scarcity, expense, and impracticality of progeny testing. However, in some special circumstances, e.g., accidental exposure of humans to potential mutagens, rhesus monkeys or other primates may provide a superior animal model to help to cope with a difficult public health situation. Using the testis as a target organ we found that when primary spermatocytes were treated in pre-leptotene stage with 1 mg mitomycin C/kg body weight, the frequency of exchanges, fragments, sex-chromosome and autosomal univalents increased significantly at diakinesis-metaphase I. This response was absent in cells treated during diplotene, late pachytene or during spermatogonial stages. We suggested that animals should be evaluated not only for genetic toxicology parameters, but also toxicologically, histologically, behaviorally, for carcinogenesis and seminal cytology. Whenever possible, the animals should be recycled.

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