Use of molecular markers to study the effects of environmental impacts on genetic diversity in brown bullhead (Ameirus nebulosus) populations.

Conservation biology needs sound biological information in order to maintain biological diversity in the face of the current rate of loss. An important component of the information needed is the level of genetic diversity within and between populations, especially for those species faced with exposure to environmental stressors. We applied multilocus DNA profile analysis (highly variable number tandem repeats [HVNTR] and randomly amplified polymorphic DNA [RAPD] techniques) and allozyme analysis to test whether individuals from historically degraded sites display levels of genetic diversity different from individuals taken from reference sites. Four Lake Erie tributaries, two impacted and two reference sites, were the sources of brown bullhead (Ameiurus nebulosus) samples. Pairwise comparison of the sampled populations demonstrated an association of decreased genetic diversity with exposure of brown bullhead to stressors using both RAPD and HVNTR analysis.

Development of DNA-based microsatellite marker technology for studies of genetic diversity in stressor impacted populations.

In recent years microsatellite marker applications in various disciplines increased rapidly. Microsatellites are characterized as independent and highly variable markers most noted for their usefulness and popularity among researchers. Microsatellites can be constructed in-house from a partial genomic library, ordered from a vendor, or "borrowed" from a species that is closely related to the target species. Microsatellite application in natural populations can reveal present as well as historical impacts of stressors to particular population.

Vitellogenin gene transcription: a relative quantitative exposure indicator of environmental estrogens.

We report the development of a quantifiable exposure indicator for measuring the presence of environmental estrogens in aquatic systems. Synthetic oligonucleotides, designed specifically for the vitellogenin gene (Vg) transcription product, were used in a reverse transcription-polymerase chain reaction (RT-PCR) protocol. This extremely sensitive and rapid method was able to detect vitellogenin gene transcription in male common carp (Cyprinus carpio) injected with 17beta-estradiol. Sequence analysis of the induced mRNA product confirmed a vitellogenin gene transcript with homology to rainbow trout and fathead minnow vitellogenin cDNA sequences. Relative levels of vitellogenin gene induction among individuals were quantified by incorporating 18S ribosomal RNA universal primers and Competimers in a PCR multiplex reaction with primers for vitellogenin. This method is more sensitive than current protocols, such as mortality, visible signs of stress, or other techniques that look for unscheduled gene expression, because it measures the appearance of primary transcripts at the nanogram level. In addition, this procedure does not sacrifice accuracy or reliability, even though the exposure to estrogen is within 1 d. This research will support the construction of regional stressor profiles, thereby providing a method for comparative environmental exposure assessment. It may also provide an in vivo screening method for potential endocrine-disrupting compounds.

Quality of assurance considerations for use of the Fluorlmager SI and fragmeNT analysis software.

The Fluorlmager SI (FSI) from Molecular Dynamics is one of several scanning instruments available for the detection of fluorescent emissions associated with DNA samples in a variety of matrices (agarose and polyacrylamide gels, membranes and microplates). In our laboratory, we measured the electrophoretic mobility of randomly amplified polymorphic DNA (RAPD) fragments stained with ethidium bromide in agarose using the FSI to scan gels and the associated Molecular Dynamics software (ImageQuaNT, and FragmeNT Analysis) for analysis. Initial scans and analyses resulted in inconsistent band detection across the same gel and across several scans of the same gel. To determine the best types of calibration for the instrument, several factors were considered and then evaluated. Tests of calibration acceptability were also evaluated. Band detection by FragmeNT Analysis was improved following optimization of matrices and parameters used in calibration and experimental scans. In addition, use of software templates for analysis and modifications in the staining procedure, which have resulted in decreased instrument associated variance, are discussed.

Genetic diversity of Typha latifolia (Typhaceae) and the impact of pollutants examined with tandem-repetitive DNA probes.

Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation were to determine whether: (1) the previously reported almost complete lack of polymorphism at allozyme loci in this species was indicative of a reduced amount of genetic diversity at VNTR loci as well; (2) VNTR markers were informative about possible clonal propagation; and (3) significant differences in genetic structure of sampling sites were associated with differences in environmental levels of pollutants at those sites. Previously, widespread sampling across the eastern United States, surveying across ten allozyme loci, has detected only two genotypes, involving a difference at a single locus, among 104 populations. In this study, the amount of genetic diversity detected at VNTR loci: (1) among ramets (N = 40; 40 genotypes detected) collected at ∼8-km intervals along a 320-km transect; (2) among ramets (N = 220; 117 genotypes detected) from five study sites separated by 50-3000 m; and (3) even among ramets within each study site [N = 44 per site; from 13 to 34 genotypes detected per site (270 m(2))] exceeds that previously found in those more geographically widespread allozyme surveys. Among the 260 ramets analyzed here, the mean number of bands scored per individual was 48.61 (SD = 2.80). Mean genetic similarity among ramets collected along the 320-km transect was 0.91, which was within the range of mean genetic similarity within the five study sites (range: 0.89-0.95). Among the five study sites, 61% of the samples analyzed appeared to be clonal ramets, with up to 12 clones detected for 44 ramets sampled within a site. Clones grew intermingled and ranged up to 39 m in extent. Permutation tests of genetic similarity revealed significant genetic differentiation between each of the five study sites. Consistent with the previous allozyme studies, T. latifolia was characterized by extremely low genetic variation relative to levels of polymorphism detected at VNTR loci in other plant species. Estimated heterozygosity among ramets along the 320-km transect ranged from 0.11 to 0.13, while that within the five study sites ranged from 0.05 to 0.12. Estimates of F(st) (0.32-0.41) also indicated considerable genetic subdivision among these stands. Significantly higher genetic diversity was detected at the two study sites that chemistry and toxicity data indicate to be the most severely impacted by pollutants. Although this correlation does not establish cause and effect, the results of this study indicate that the analysis of genetic diversity at VNTR loci may be a useful tool for monitoring anthropogenic-induced changes in the genetic structure of natural populations of plants.

Male reproductive effects of solvent and fuel exposure during aircraft maintenance.

Few studies have addressed the effects of mixed, low-level exposures to complex mixtures on a man's reproductive potential. In this prospective study, each subject was evaluated before first exposure and at 15 and 30 weeks after exposures had begun. A total of 50 men working on aircraft maintenance at an Air Force installation were included in the study. In addition, eight unexposed men were concurrently sampled. Industrial hygiene (IH) sampling and expired breath samples were collected for jet fuel as measured by total napthas, benzene--a component of jet fuel, 1,1,1-trichloroethane, methyl ethyl ketone, xylenes, toluene, and methylene chloride. Sperm production, structure, and function (sperm concentration, sperm motion, viability, morphology, morphometrics, and stability of sperm chromatin) were evaluated. Exposures were low. All mean IH measures were below 6 ppm, which is less than 10% of the Occupational Safety and Health Administration standard for all chemicals except benzene. Sheet metal workers had the highest mean breath levels for both total solvents (24 ppb) and fuels (28.3 ppb). For most sperm measures, mean values remained in the normal range throughout the 30 weeks of exposure. When jobs were analyzed by exposure groups, some adverse changes were observed. The paint shop group had a significant decline in motility of 19.5% at 30 weeks. Internal dose measures, however, did not show a significant association with spermatogenic changes.

Hypothesis testing with the similarity index.

Multilocus DNA fingerprinting methods have been used extensively to address genetic issues in wildlife populations. Hypotheses concerning population subdivision and differing levels of diversity can be addressed through the use of the similarity index (S), a band-sharing coefficient, and many researchers construct hypothesis tests with S based on the work of Lynch. It is shown in the present study, through mathematical analysis and through simulations, that estimates of the variance of a mean S based on Lynch's work are downwardly biased. An unbiased alternative is presented and mathematically justified. It is shown further, however, that even when the bias in Lynch's estimator is corrected, the estimator is highly imprecise compared with estimates based on an alternative approach such as 'parametric bootstrapping' of allele frequencies. Also discussed are permutation tests and their construction given the interdependence of Ss which share individuals. A simulation illustrates how some published misuses of these tests can lead to incorrect conclusions in hypothesis testing.

Effect of cryopreservation and theophylline on motility characteristics of lake sturgeon (Acipenser fulvescens) spermatozoa.

Computer-assisted motility analysis (CASA) was used to evaluate the effect of cryopreservation and theophylline treatment on sperm motility of lake sturgeon (Acipenser fulvescens ). Motility was recorded at 0 and 5 min postactivation. The effect of cryopreservation on sperm acrosin-like activity was also measured. Cryopreservation led to a decline in the percentage of motile spermatozoa, while other parameters of sperm motion, curvilinear and straight line velocities, linearity and amplitude of lateral head displacement were unchanged. Reductions in straight line velocity observed with fresh and cryopreserved spermatozoa and in linearity with cryopreserved spermatozoa 5 min postactivation were not seen in the presence of 5 mM theophylline at this time point. Frozen-thawed spermatozoa retained acrosin-like activity, and it correlated with the percentage of post-thaw motility (r = 0.95 and r = 0.90, P < 0.05, for 0 and 5 min post-activation time, respectively).

Adverse male reproductive effects following subchronic exposure of rats to sodium dichloroacetate.

Dichloroacetate (DCA) activates the pyruvate dehydrogenase complex enhancing carbohydrate and lactate utilization in animals. As a result it is used clinically in the treatment of acute lactic acidosis and has therapeutic potential in the treatment of stroke. Adverse effects of chronic DCA treatment include polyneuropathy and testicular degeneration. Since DCA is a principal product of the aqueous chlorination of fulvic acids concern has arisen regarding the agent's impact on environmental health. We treated male Long-Evans rats with 0, 31.25, 62.5, or 125 mg DCA/kg/day by oral gavage for 10 weeks. Compared to controls, preputial gland and epididymis weights were reduced at 31.25 mg/kg, body and liver weights at 62.5 mg/kg, and accessory organ weights at 125 mg/kg. Epididymal sperm counts were reduced and sperm morphology was impacted at the 62.5 and 125 mg/kg doses levels. Histologic examination of the testis and epididymis revealed inhibited spermiation in testes at the 125 mg/kg dose level. Computer-assisted sperm motion analysis revealed reductions in percentage motile sperm, curvilinear and straight-line velocity, linearity, and amplitude of lateral head displacement at both the 62.5 and the 125 mg/kg dose levels. In the assessment of fertility after an overnight mating, the number of viable implants on Day 14 of gestation was decreased only in the highest dose group. These studies demonstrate adverse effects of NaDCA treatment on the rat male reproductive system, primarily on the accessory organs and sperm within them at lower doses (31.25 and 62.5 mg/kg), and on the testis at the highest dose (125 mg/kg).

Effects of three male reproductive toxicants on rat cauda epididymal sperm motion.

The sensitivity of the CellSoft computer-assisted sperm analysis (CASA) system to detect changes in rat sperm motion was evaluated. CASA motion endpoints were measured in cauda epididymal sperm from Long-Evans rats treated with each of three known male reproductive toxicants reported to affect the epididymis and epididymal sperm motility: alpha-chlorohydrin, ornidazole, and trimethylphosphate. Significant changes in endpoints describing sperm swimming vigor (curvilinear velocity and straight-line velocity) and pattern (linearity and amplitude of lateral head displacement) were observed for rats dosed with each agent when evaluations included mean values and other statistical parameters (i.e., percentiles and distributional shape). alpha-Chlorohydrin (ACH) treatment (10 mg/kg/day; 8 days) resulted in reductions in the mean percentage of motile sperm, curvilinear velocity (VCL), straight-line velocity (VSL), lateral head displacement (ALH), and linearity (LIN). Treatment with ornidazole (ONZ) (200 mg/kg/day/14 days) reduced the percentage of motile sperm. Mean VCL, VSL, and ALH were reduced by 400 mg ONZ/kg/day treatment. Trimethylphosphate (TMP) treatment led to (a) a reduction in the 75th and 90th percentiles for ALH (100 mg TMP/kg/day; 5 days) (P < or = 0.04), (b) a reduction in VCL, VSL, and ALH (250 mg TMP/kg/day), (c) a reduction in the percentage of motile cells and in the 10th and 25th percentiles for VSL (600 mg TMP/kg/day), and (d) increases in the 90th percentile for VSL, in the mean, 75th, and 90th percentiles for VCL, and in the 75th and 90th percentiles for ALH (600 mg TMP/kg/day). The general utility of these analytic approaches in reproductive toxicology studies was demonstrated in the observations of effects at or below dose levels previously reported.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

Sources of variation in the computer-assisted motion analysis of rat epididymal sperm.

Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.

Effects of chlorine dioxide on the developing rat brain.

Male and female Long-Evans rat pups, exposed to an oral dose of 14 mg chlorine dioxide (CIO2)/kg/d (postnatal d 1-20), were examined for effects on brain development and for changes in thyroid activity. Body weight reductions were observed on postnatal (pn) d 11, 21, and 35. Forebrain weight and protein content were decreased on pnd 21 and 35, as were the DNA content on d 35 and the number of dendritic spines on cerebral cortical pyramidal cells, a marker for synapse formation. Otherwise, cell proliferation in the forebrain, cerebellum, and olfactory bulbs was normal, as were migration and aggregation of neuronal cells in three areas of the cerebral cortex. Histopathology of the forebrain, cerebellum, and brainstem showed no gross lesions, loss of myelin, or change in the cells staining positive for Nissl substance. Serum T3 and T4 levels, as well as hepatic mitochondrial alpha-glycerophosphate dehydrogenase activity, were unchanged by CIO2 treatment. The results indicated that CIO2 may have central neurotoxic potential. No underlying antithyroid activity was evident.

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations.

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.

Effects of epichlorohydrin on male and female reproduction in Long-Evans rats.

Male and female Long-Evans rats were treated with epichlorohydrin (ECH) by oral gavage (males: 12.5, 25, and 50 mg/kg/day; females: 25, 50, and 100 mg/kg/day) for 21 and 14 days, respectively, prior to mating trials with untreated animals. Treated females were further dosed until delivery. Fertility was assayed in the high-dose males only and was found to be totally impaired. No measured parameters of female reproduction were changed relative to controls. Treated males showed normal copulatory behavior. Sperm morphology and percentage motile sperm were not statistically different from control values in both ejaculated and cauda epididymal samples from ECH-treated animals. The number of sperm in ejaculates was normal while cauda epididymal sperm count was slightly decreased in males at the 50 mg ECH/kg dose level. Mean curvilinear velocity, straight-line velocity, and amplitude of lateral head displacement of cauda epididymal sperm were significantly reduced by ECH at 12.5 mg/kg/day and above. Sperm track linearity was also reduced, but only at 50 mg/kg/day. Beat/cross frequency of sperm was significantly increased at 12.5 mg/kg/day and above. All of the above sperm motion parameters showed dose-dependent trends. These effects are consistent with the spermatozoal metabolic lesions reported for alpha-chlorohydrin, a metabolite of ECH.

Effects of divalent cations on acetylcholine release from digitonin-permeabilized rat cortical synaptosomes.

A preparation of rat brain synaptosomes, partially permeabilized by the cholesterol-specific detergent digitonin, was developed to study the effects of the heavy metals Pb+2 and Cd+2 and of other divalent cations on acetylcholine release from nerve terminals. Consistent with the cation specificities of the transmitter release process in intact nerve terminals, 100 microM free Ba+2, Sr+2 and Ca+2 each induced a release of acetylcholine (ACh) while Ni+2, Co+2 and Mn+2 did not. 100 microM Pb+2 and Cd+2 also induced a release of ACh from the permeabilized synaptosomes. This suggests that, similar to Ba+2 and Sr+2, both Pb+2 and Cd+2 may induce transmitter release through a direct action on the transmitter release apparatus.