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M L Meistrich

Publications and source records attributed to M L Meistrich.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of the rat spermatid basic nuclear protein TP4.

Following elongation of spermatids in mammals, the histones are replaced by a set of basic nuclear transition proteins; in the rat there are four, named TP1-TP4. Of these, TP1 and TP2 are well characterized. Here we report the purification to homogeneity of TP4 from rat spermatids. It is a low molecular mass (about 13-20 kDa) basic protein with arginine and lysine constituting 24 mol % and histidine 2.2 mol %. Its 25 N-terminal amino acids were sequenced, and no sequence homologies with any known protein were found. Polyclonal antibodies raised against it in rabbit did not cross-react with other transition proteins, protamines, or histones. The presence of TP4 during sperm development was monitored by cell separation studies. No TP4 was detected in round spermatids, and along with TP1 and TP2, it is present in step 13-15 spermatids and its amount decreased in steps 16-19. Trace amounts of TP4 were also detected in epididymal sperm. A possible role for TP4 in spermatid and sperm chromatin structure is discussed.

Amino Acid Sequence↗
PubMed1992

Impact of cyclophosphamide on long-term reduction in sperm count in men treated with combination chemotherapy for Ewing and soft tissue sarcomas.

BACKGROUND: Treatment of cancer with multiple-drug chemotherapy regimens or radiation therapy can cause either temporary azoospermia of various durations or permanent azoospermia in young men. METHODS: To identify which drugs in which doses contribute to long-term or permanent azoospermia, semen analyses were done on patients with Ewing and soft tissue sarcomas before, during, and after treatment with either CYADIC (cyclophosphamide, doxorubicin, and dacarbazine), or CYVADIC (vincristine added to CYADIC). Some patients also received other drugs or radiation therapy. RESULTS: From pretreatment levels that were similar to those of control subjects, sperm production declined to azoospermia within 4 months of treatment. Sperm production returned in some patients after treatment; 40% of men recovered to normospermic levels by 5 years after treatment. Few patients showed continued recovery of sperm production after that time. The cumulative dose of cyclophosphamide was the most significant determinant of recovery to normospermic levels; approximately 70% of those who had received doses less than 7.5 g/m2 (median, 4.1 g/m2) recovered, but only 10% recovered when doses exceeded 7.5 g/m2. CONCLUSIONS: Thus, a risk of permanent sterility is associated with the use of the CYADIC and CYVADIC regimens in young men, especially when the cumulative dose of cyclophosphamide is greater than 7.5 mg/m2.

Adult↗
PubMed1992

Highly acetylated H4 is associated with histone displacement in rat spermatids.

The presence of highly acetylated histone H4 during spermatogenesis was studied to evaluate its correlation with the events of gene transcription, histone deposition, and histone displacement. We utilized an antibody raised to a pentaacetylated synthetic peptide that preferentially recognizes highly (tetra- and tri-) acetylated forms of rat testis H4. Electrophoretic separation of histones from enriched fractions of spermatogenic cells followed by detection of these forms by staining and by immunoblotting using this antibody showed that the highly acetylated forms were limited almost exclusively to spermatids beginning at step 11 of development. Immunoflurescence also revealed a striking polarity in the progression of histone from the spermatid nucleus. Highly acetylated H4 was displaced from the anterior to the caudal portion of the spermatid nucleus during steps 11 and 12, along with other histones, prior to their displacement by transition proteins. Thus, while monoacetylated and low levels of diacetylated forms of H4 were associated with stages at which histone deposition and transcription occur, the more highly acetylated forms appeared in high levels only at the stage at which histone displacement occurs.

Acetylation↗
PubMed1992

A method for quantitative assessment of reproductive risks to the human male.

There is a need for quantitative assessment of reproductive risks following human exposure to toxic agents. Current methods only estimate exposure levels that are unlikely to produce appreciable risk; they do not address the magnitude of risk. The quantitative reproductive risk estimation (QRRE) approach presented here enables calculation of the increased incidence of infertility in a human population resulting from exposure of males to a toxic agent. The four steps involve (A) obtaining a dose-response curve in experimental animals for the effect of a toxicant on a measure of reproductive function; (B) applying an interspecies extrapolation factor (IEF) derived for a similar toxicant to relate doses in animals and humans that produce equivalent reproductive toxicity; (C) using human exposure and uptake levels, along with the IEF and the dose-response curve, to calculate impairment of the measure in humans; and (D) computing the increase in human infertility caused by that impairment. The QRRE method is used to compute reproductive risks from dibromochloropropane exposure.

Animals↗
PubMed1992

Acceleration of late radiation damage of the kidney by unilateral nephrectomy.

Both increased proliferation as measured by labeling index and the appearance of abnormally large nuclei in renal proximal tubule cells, which have been observed in mouse kidneys after irradiation, were enhanced by subsequent unilateral nephrectomy. Nephrectomy alone induced only a transient increase in labeling index, lasting less than 1 month, whereas nephrectomy after irradiation induced an increase above that of the irradiated kidneys without nephrectomy lasting as long as 9 months. The incidence of large nuclei in kidneys from mice unilaterally nephrectomized 1 week after irradiation showed a rapid increase with time, peaking at 4.6% at 6 months, compared to more gradual increases with peaks at about 4.0% at 9 or 12 months in irradiated kidneys without nephrectomy or those in which nephrectomy was done prior to irradiation. This result demonstrates that nephrectomy after irradiation accelerates the appearance of this indicator of radiation damage, rather than enhancing the maximum amount of damage. Unilateral nephrectomy after irradiation also increased kidney damage 9 months later, as indicated by kidney weight loss and increased blood urea nitrogen. These results are consistent with our model for radiation damage of the kidney in which radiation induces cell proliferation and the appearance of reproductively dead, large nuclear cells that are lost at subsequent attempts to divide; the acceleration of radiation damage by unilateral nephrectomy performed after radiation could very well be a result of nephrectomy-induced enhancement in the proliferation of proximal tubule cells.

Animals↗
PubMed1992

Spermatogenesis in retinol-deficient rats maintained on retinoic acid.

Rats maintained on a diet deficient in retinol and retinoic acid were given a diet containing retinoic acid for 21-29 days after the start of weight loss. The testes of four of these rats were studied. Spermatogonia of all types were observed, though in lower numbers than in controls, and their mitotic activity was normal. Normal preleptotene spermatocytes were encountered, but no normal spermatocytes in further stages of development were seen. Pale cells that appeared to be in prophase were observed. It was concluded that, in retinol-deficient rats maintained on retinoic acid, the spermatogonial population is qualitatively normal, but quantitatively subnormal, while spermatocyte development is qualitatively and quantitatively abnormal. No evidence of spermatogonial arrest or any other form of synchronization was found in testes of these rats, but when the remaining rats were injected with retinol, the seminiferous epithelium did show stage synchronization at 36 and 128 days after the injection.

Animals↗
PubMed1991

Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents.

Structural features of the mouse and rat manchette and the role of the manchette in shaping the spermatid nucleus were investigated. Rod-like elements about 10 nm in diameter and 40-70 nm in length were seen linking the innermost microtubules of the manchette and the outer leaflet of the nuclear envelope in step 8 through step 11 rat and mouse spermatids that either had been routinely fixed for electron microscopy or had been isolated and detergent extracted. Rod-like linkers were also seen joining the nuclear ring to the plasma membrane and nuclear envelope. These linkers may ensure that under normal conditions the manchette remains in a defined position relative to these membranous components. A variety of compounds (taxol, cytoxan, and 5-fluorouracil) were found to perturb the manchette and to affect nuclear shaping. In addition, sys and azh mutant mice were used to determine the consequences of defective manchette formation. These genetic conditions and chemical treatments either produced manchettes that were not in their normal position (azh, sys, and taxol) and/or caused the manchette to appear abnormal (azh, sys, cytoxan, 5-fluorouracil, and taxol), and all resulted in a deformation of the step 9-11 spermatid nucleus. In all instances where the manchette was present, either in normal or ectopic locations, the sectioned nuclear envelope was parallel to the long axis of the microtubules of the manchette. In general, areas of the nuclear envelope where the manchette was not present, or where it was expected to be present but was not, were rounded (normal animals, sys, cytoxan). In addition, there are indications using certain compounds (cytoxan and 5-fluorouracil) as well as in the azh and sys mouse that the manchette may exert pressure to deform the nucleus. It is suggested that the rod-like linkages of the manchette ensure that the nuclear envelope remains at a constant distance from the manchette microtubules and that this is a major factor acting to impart nuclear shape changes on a region of the head caudal to the acrosome during the early elongation phase of spermiogenesis. The manchette microtubules, which are also known to be linked together, may act as a scaffold to deform this part of the nucleus from its spherical shape, perhaps in concert with forces initiated by other structural elements. Evidence from sys animals indicates that structural elements, such as the acrosomal complex over the anterior head (acrosome-actin-nuclear envelope), may affect nuclear shaping over the acrosome-covered portion of the spermatid head.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells.

Various methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well-defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling. The relative movement (RM) of BrdUrd-labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, v (a function of the fraction of BrdUrd-labeled divided and the fraction of BrdUrd-labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time-point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.

Animals↗
PubMed1991

Stage-synchronized seminiferous epithelium in rats after manipulation of retinol levels.

Optimal conditions for obtaining stage-synchronization of the seminiferous epithelium were investigated. In this study, 147 rats were subjected to protocols in which vitamin A deficiency was induced by feeding a diet without retinol (R-ol) or retinoic acid (RA), followed by maintenance on a diet containing RA and supplementation of R-ol by injection and diet. An acceptable degree of stage synchronization and recovery of the seminiferous epithelium was observed in 90 (61%) of the 147 rats. The effects on synchrony of variations in the protocol, including the degree of deficiency before RA maintenance, the dose and duration of RA maintenance, and the manner of injection of R-ol, were tested. Initiation of maintenance on RA when a medium degree of deficiency was achieved (4-12 g of weight loss, 3-6 days without growth) resulted in a more reliable (80% of the rats) induction of synchrony than did initiation of maintenance on RA at either a less (70% synchronized rats) or more severe (50-60% synchronized rats) deficiency. Maintenance on food containing 10 mg/kg RA gave better and more reliable synchrony (70%) than maintenance on food containing 5 mg/kg RA (less than 40%). Although the duration of this maintenance did not influence the degree of synchrony, the reliability was lower when maintenance was continued for a month or more (54%). During the interval from 33 to 128 days after resupplementation, the degree of synchronization decreased, as did the predictability of the stages, while the restoration of spermatogenesis increased. Linear regression, performed on the location of the median point of synchronization, indicated that spermatogenesis progressed at a rate of 12.4 days per cycle. The median stage of synchronization, predicted by this regression line, differed by an average of 8% of the cycle from the actual location in individual rats. Extrapolation of the regression line indicated that spermatogenesis was reinitiated in mid-to-late stage VII.

Administration, Oral↗
PubMed1991

Measuring cell proliferation by relative movement. I. Introduction and in vitro studies.

This paper presents two new ways of analysing data which may be obtained from pulse labelling a population of cells with bromodeoxyuridine and analysing that population as a function of time with bivariate flow cytometry. The progression of cells is measured by the change in position in the cell cycle, as shown by a change in the mean DNA content of the labelled and unlabelled cells. The particular measures of the mean DNA content used are extensions of the relative movement of the labelled undivided cells, RMlu(t), which was introduced by Begg and co-workers to measure the DNA synthesis time, TS. In general, the relative movement is defined as the mean DNA fluorescence of a population of cells less the DNA fluorescence of the cells in G1 and divided by the difference in DNA fluorescence of the cells in G2 + M and G1. In this paper we examine the relative movements of all the labelled cells and all of the unlabelled cells, denoted RML(t) and RMU(t) respectively. It is found that RML(t) and RMU(t) exhibit clear cyclic behaviour and distinguishable characteristics which depend directly on the transit times (T) of the cell cycle phases, i.e. TG1, TS and TG2 + M. Furthermore, the peak heights of the RMU(t) curve are shown to depend strongly on the growth fraction of the population under consideration. A theoretical treatment of the curves so obtained is presented, and is shown to yield values in close agreement with those from other methods for measuring these transit times and a lower limit to values for the growth fraction of Chinese hamster ovary cells grown in vitro.

Animals↗
PubMed1990

Low levels of chromosomal mutations in germ cells derived from doxorubicin-treated stem spermatogonia in the mouse.

The mutagenic effects of doxorubicin (Adriamycin, ADR) on mouse spermatogonial stem cells were examined by analysis of spermatocyte chromosomes and of dominant lethality transmitted through the spermatozoa. The effects of ADR on mutations, cytotoxicity, and sperm head abnormalities were compared with those of radiation. The cytotoxic effect of 6 Gy of gamma-radiation on stem spermatogonia was equivalent to about 4-5 mg ADR/kg. Chromosomal translocations were observed in 0.6% of the spermatocytes of mice treated with ADR (2-6 mg/kg). In contrast, 6 Gy of radiation induced translocations in 11.1% of spermatocytes. No increase in dominant lethality was observed after treatment with ADR at doses up to 6 mg/kg, while the frequency after 6 Gy of radiation was 3.6%. Based on these results, ADR would be expected to be only a weak inducer of balanced chromosomal rearrangements. Because ADR at 4.5 mg/kg was much weaker than 6 Gy of gamma-radiation at inducing chromosomal translocations, but just as effective at inducing sperm head abnormalities, the level of sperm head abnormalities is not indicative of balanced chromosomal rearrangements induced in stem spermatogonia by cytotoxic agents.

Animals↗
PubMed1990

Abnormal manchette development in spermatids of azh/azh mutant mice.

A study of manchette development during spermiogenesis in azh/azh mutant mice was carried out by thin-section transmission electron microscopy with the goal of determining which of the initial steps in spermatid development are aberrant. In the homozygous mutant, spermatogenesis was quantitatively normal; but 100% of the sperm nuclei produced had abnormal shapes. The first defect, observed in steps 8-9, was the abnormal positioning of many manchette microtubules. These microtubules were directed towards regions of the plasma membrane not normally associated with manchette formation, in addition to being located at the caudal rim of the acrosome in the normal region of manchette formation. At steps 10-12, sheets of manchette microtubules were often in ectopic positions along the plasma membrane, rather than in association with the nuclear membrane as well. The fine structural appearance of the manchette was generally normal; the defect appeared to be in its positioning within the cell. In many step 8-10 spermatids nuclear invaginations and evaginations were observed, always associated with irregularities in the position of some of the manchette microtubules; these illustrate the capacity of manchette microtubules to deform nuclear shape. The nuclear irregularities remained throughout spermiogenesis. These observations are consistent with the hypothesis that the manchette is involved in at least some aspects of sperm nuclear shaping and that the improper positioning of manchette formation is a likely candidate for the primary abnormality resulting from a defective allele at the azh locus.

Animals↗
PubMed1990

Improved method for computing potential doubling time from flow cytometric data.

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.

Animals↗
PubMed1990

The accuracy of fine needle aspiration biopsy for flow cytometric determination of tumor DNA content.

The use of fine needle aspiration (FNA) to obtain a diagnosis of malignancy is established in the practice of oncology, but there is little information on its accuracy in sampling tumor DNA content. We therefore compared flow cytometric DNA data obtained from FNA-derived samples with that obtained after digestion of the same murine tumor from which the aspirates had been taken. Fifteen female C3Hf/Kam mice were implanted with MCA-29 tumor cells from the same source tumor. MCA-29 is a multiploid mammary adenocarcinoma with two aneuploid populations (DNA Index of A = 1.67, B = 1.89). The tumors were grown to a mean size of 8.6 mm. After sacrifice, three FNAs were performed on each tumor, following which the whole tumor (WT) was excised and homogenized. All FNA and WT samples were digested with 0.04% pepsin and the nuclei stained with propidium iodide in preparation for flow cytometry. DNA histograms of the aspirates were compared with the corresponding WT histograms. Any single FNA detected population A in all (100%) cases and detected the less prominent population B in 94.3% of instances. Any single FNA was able to detect the same populations that were present in the whole tumor in 95.4% of cases, while the set of three aspirates matched the corresponding WT in 100% of cases. We conclude that FNA DNA histograms are accurate for the assessment of ploidy, but that in order to ensure detection of all tumor populations present, multiple aspirates are needed.

Animals↗
PubMed1990

Symplastic spermatids (sys): a recessive insertional mutation in mice causing a defect in spermatogenesis.

A line of transgenic mice that carries an insertional mutation in a gene essential for spermatogenesis is described. Males homozygous for the transgenic insert are sterile, while female homozygotes and both male and female heterozygotes exhibit normal fertility. Developing spermatids in homozygous males form prominent abnormal multinucleated syncytia (symplasts) and do not complete maturation. In addition, abnormal cytoplasmic vacuolation is commonly seen in Sertoli cells. One flank of the transgenic integration site within the genome has been cloned and used to show linkage between homozygosity for the transgene and the mutant phenotype. The flank maps to mouse chromosome 14 approximately 4 centimorgans proximal to the gene encoding esterase-10 (Es-10). As no other gene that is known to be essential for spermatogenesis has been mapped to this region of the genome and as the mutant phenotype is unique, the transgenic insert appears to affect a previously unidentified gene. We have named the mutation "symplastic spermatids" (sys).

Alleles↗
PubMed1990

A method for quantifying synchrony in testes of rats treated with vitamin A deprivation and readministration.

Using a variation of a previously published method for manipulating vitamin A levels, we obtained synchronized rat testes and determined the frequency of stages of the seminiferous epithelium in each rat. In this study, we have demonstrated a method for quantitative analysis of the synchrony. The degree of synchronization was expressed as a fraction of the cycle of the seminiferous epithelium, and thus in terms not influenced by the different durations of the stages of this cycle. The median stage about which the tubules were synchronized was calculated. This method may be used to compare the effects of different synchronizing treatments, which may be subtle, and to study various aspects of spermatogenesis in the synchronized testes. For example, the duration of the cycle of the seminiferous epithelium in synchronized testes is estimated to be 12.5 days.

Animals↗
PubMed1990

Probability of self-renewing divisions of spermatogonial stem cells in colonies, formed after fission neutron irradiation.

Repopulating spermatogenic colonies, found in the seminiferous epithelium after irradiation with fast-fission neutrons, were studied to determine the chance that a stem cell Asingle (As) spermatogonium would complete a self-renewing division (P). Mathematical formulas originally derived for such studies in haemopoietic colonies were employed, and a method specifically aimed at spermatogenic colonies was developed. The results showed that during the first division after irradiation, P is close to 1.0. P decreases in later generations, but remains 0.7 or higher up to the 4th or 5th divisions. The mean value for P was over 0.8, which is higher than the value of 0.6-0.7 found for stem cells in haemopoietic colonies.

Animals↗