Flow cytometry of DNA in mouse sperm and testis nuclei

Meistrich, Marvin L.; Lake, Suzanne; Steinmetz, L. L.; Gledhill, Barton L.

doi:10.1016/0027-5107(78)90109-4

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

1978

DOI: 10.1016/0027-5107(78)90109-4

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Flow cytometry of DNA in mouse sperm and testis nuclei

Marvin L. Meistrich

Suzanne Lake

L. L. Steinmetz

et al.

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1982

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Cited by 40 publications

(19 citation statements)

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“…This test is relatively rapid and is considered very useful in the assessment of potentially toxic chemicals, since 100% of known mutagens tested were correctly identified as positives (44). Thus, demonstration of elevated levels of sperm abnormalities following exposure to a physical or chemical agent raises the possibility that the agent is inducing mutations in the germ cells (33,39).Recently, we have developed a flow cytometric (FCM) assay of sperm chromatin structure (8) to determine the ~ Address reprint requests to Donald …”

mentioning

confidence: 99%

“…This test is relatively rapid and is considered very useful in the assessment of potentially toxic chemicals, since 100% of known mutagens tested were correctly identified as positives (44). Thus, demonstration of elevated levels of sperm abnormalities following exposure to a physical or chemical agent raises the possibility that the agent is inducing mutations in the germ cells (33,39). inherent stability of sperm nuclear DNA to thermal denaturation in situ and have found this assay to be useful in measuring effects of disease, diet, chemotherapy, and other factors on sperm nuclear chromatin structure (7,10,12).…”

mentioning

confidence: 99%

“…The work reported here on effects of ethylnitrosourea (ENU) and other ongoing research efforts (11) are testing the hypothesis that flow cytometric analysis of sperm samples from mice exposed to mutagenic and carcinogenic chemicals by the same protocol of Wyrobek and Bruce can rapidly detect chemically induced sperm defects and provide additional information not available by the sperm morphology test. Our two-parameter flow cytometric analysis for ratios of testicular cell types (14) present after chemical exposure is much more rapid than screening histology sections by light microscopy and provides more information than the one parameter FCM analysis more commonly used (33).…”

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confidence: 99%

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Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

et al. 1985

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The effects of the mutagenic agent ethylnitrosourea ( E m on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mgkg body weight x 5 days and were sacrificed 28 days later. Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry. Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO. Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50%. Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals. Flow cytometric analysis of both heat-stressed and non-heatstressed nuclei showed a relationship between dosage and the coefficient of variation of at {redked + green fluorescence)] measurements of A0 stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU. Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of singlestranded DNA induced by heat or acid treatment of chemically altered chromatin structure. The lowest daily dosage used (5 mgkg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as at. This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test.Key terms: Flow cytometry, sperm head morphology, toxicology, testis, chromatin structureThe production of sperm represents one of the most dramatic examples of cellular proliferation and differentiation in any mammalian organ system. Although many biological parameters have been related to male fertility, sperm morphology is frequently used to assay testicular function. Sperm head morphology, consistent with nuclear morphology, is genotype specific (28), and is directed by genes on both the autosomes and sex chromosomes (2,411. Heritable changes in sperm head morphology can be induced by radiation and alkylating agents and followed quantitatively through successive generations (22,38,40). Wyrobek and Bruce (42,43) developed a mouse model assay system to study the mutagenic andor carcinogenic potential of chemicals using sperm head morphology as an end point. This test is relatively rapid and is considered very useful in the assessment of potentially toxic chemicals, since 100% of known mutagens tested were correctly identified as positives (44). Thus, demonstration of elevated levels of sperm abnormalities following exposure to a physical or chemical agent raises the possibility that the agent is i...

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mentioning

confidence: 99%

“…This test is relatively rapid and is considered very useful in the assessment of potentially toxic chemicals, since 100% of known mutagens tested were correctly identified as positives (44). Thus, demonstration of elevated levels of sperm abnormalities following exposure to a physical or chemical agent raises the possibility that the agent is inducing mutations in the germ cells (33,39). inherent stability of sperm nuclear DNA to thermal denaturation in situ and have found this assay to be useful in measuring effects of disease, diet, chemotherapy, and other factors on sperm nuclear chromatin structure (7,10,12).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

et al. 1985

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“…Flow cytometry has been used to analyze mouse testicular cells from minced testes, epididymis, and cell susthese studies, staining was performed with acriflavinFeulgen (17,18), acridine orange (4,6,7,10), or Hoechst 33342 (9). Relative proportions of testicular cells were quantified during maturation (10).…”

mentioning

confidence: 99%

DNA flow cytometric analysis of mouse seminiferous epithelium

Toppari¹,

Bishop²,

Parker³

et al. 1988

Cytometry

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In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide.DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XI1 showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.

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“…This pilot study presents some results and tries to establish a correlation between changes in the cell population of the testicular tissue and the patient's clinical diseases. The experimental research on animal testes (8,10) can not replace such investigations since the animals are either healthy or suffering from a limited number of inducible diseases.…”

mentioning

confidence: 99%

Flow cytometry of human testicular tissue

et al. 1982

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Flow cytometry of testicular tissue from autopsies showed differences in the proportion of substained spermatozoa (1 CA), spermatids (1 CB), diploid 2C nuclei and premeiotic spermatozoa in the 4 C class. The evaluation of the histograms was performed with computer assistance. Healthy accident cases or those delivered in forensic medicine furnished the control values. Patients with hepatic cirrhosis showed a nearly complete loss of the haploid class, while cases with fatty degeneration of the liver demonstrated a reduction. Circulatory insufficiency in A considerable amount of work has been done on human spermatozoa by flow cytometry (FCM) (4, 5, 7, 9, 11, 18, 20). Some investigations were published on testicular biopsies (3, 17, 21-23); however, the technique limits these investigations to patients with possibly malignant tumors or andrological irregularities. Cases with other diseases can only be studied with post mortem material (13, 14). This pilot study presents some results and tries to establish a correlation between changes in the cell population of the testicular tissue and the patient's clinical diseases. The experimental research on animal testes (8, 10) can not replace such investigations since the animals are either healthy or suffering from a limited number of inducible diseases. Materials and MethodsOne testis from each of 122 autopsies performed 18 to 48 hr post mortem is used for this investigation. In all but two cases histograms were obtained that could be evaluated by the computer program in spite of increased coefficients of variation (CV) which in some cases were due to the fact that human autoptic material was used. For morphologic control and histology, imprints from fresh unfixed testicular tissue are made. Stains are hematoxylin-eosin, Van Gieson,.For FCM the tissue is separated from the tunica albuginea by scraping and then minced with scissors. After treatment with 0.5% pepsin in 0.2% HCL for 5 min on a stirrer in room temperature, the suspension is filtered for the first time through a four-fold gauze mesh and either fiied in 96% ethanol, or immediately stained after 2 washings in saline. Stains are ethidiumbromide and mithramycin (1).' Presented at the VIII Conference on Analytical Cytology and Cytometry, Wentworth-by-the-Sea, New Hampshire, May 19-25, 1981. cases of acute shock resulted in an increase in the 4C values of meiotic prophase I and a decrease of spermatids. Spermatozoa were normal in these cases. After prolonged shock a nearly complete breakdown of spermatogenesis was observed. In cases of atherosclerosis an irregular degree of reduction was seen. Cancer patients showed a reduction of spermatogenesis in correlation to therapy.

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Flow cytometry of DNA in mouse sperm and testis nuclei

Cited by 40 publications

References 14 publications

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea

DNA flow cytometric analysis of mouse seminiferous epithelium

Flow cytometry of human testicular tissue

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