Gregory P. Toth

Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions

Abstract An objective analysis of the duration of motility of sperm from the lake sturgeon, Acipenser fulvescens , has been performed using computer-assisted sperm motion analysis at 200 frames/s. Motility was measured in both 1993 and 1994. The percentage of activated motile sperm and their velocities ([1993] curvilinear velocity: 313 μm/s; straight-line velocity: 129 μm/s) were unchanged after 5 min in Tris/glycine buffer, pH 9.0, with 10 mM added Na + . Qualitative, visual measurements revealed motility lasting for 30 min in this medium. The percentage of motile cells, but not the velocities of the motile cells, was reduced by the addition of potassium to the Tris/glycine buffer. The percentage of motile sperm was inhibited by 50% at 0.5 mM added K + . Elemental analysis of seminal plasma revealed the following concentrations: P (3.02 mM); K (5.78 mM); Ca (0.16 mM); Mg (0.21 mM); Na (25.6 mM); Zn (0.76 μM); and Cl (5.41 mM). Differences between two years of milt collection were not significant.

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).

Methods for assessing rat sperm motility

Computer-assisted sperm analysis (CASA) systems are becoming more widely used. With this spread of technology come more data from toxicology studies, designed to determine if treatment with putative toxicants affects sperm motion parameters. While these CASA methods provide us with more ways to evaluate toxicity and thus perhaps increase our chances of successfully protecting human health, there is also a greater likelihood that different laboratories will use different methods of collecting data on sperm motility. Different systems used with different methods in different laboratories will inevitably generate data that are difficult to compare. In a prospective attempt to address this issue of comparability and limit the problems, a group of individuals using CASA systems to analyze rat sperm motility convened to discuss methodologic issues, share data, and try to reach a consensus about methods for performing these studies. This article shares those meetings and data in the hope that common methods will enhance interlaboratory comparisons.

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 agents 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).

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.

LABORATORY METHODS FOR ASSESSING HUMAN SEMEN IN EPIDEMIOLOGIC STUDIES: A CONSENSUS REPORT

It is clear that additional methodologic work needs to be performed. Some data gaps described above are being actively investigated. Other standards were not addressed at this meeting; statistical handling of the data, differences among CASA machines, and factors to consider as potential confounders in analysis are just a few. These may be the subject of future workshops, which will also review progress made in the existing knowledge base. For now, this effort represents a first attempt to share information and to use it to encourage investigators in different laboratories to employ similar methods. In this way more direct comparisons among studies can be made, and our collective data base can be strengthened.

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

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.

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.

The path from molecular indicators of exposure to describing dynamic biological systems in an aquatic organism: microarrays and the fathead minnow.

The extent to which humans and wildlife are exposed to toxicants is an important focus of environmental research. This work has been directed toward the development of molecular indicators diagnostic for exposure to various stressors in freshwater fish. Research includes the discovery of genes, indicative of environmental exposure, in the Agencys long-established aquatic toxicological organism, the fathead minnow (Pimephales promelas). Novel cDNAs and coding sequences will be used in DNA microarray analyses for pattern identification of stressor-specific, differentially up- and down-regulated genes. The methods currently used to discover genes in this organism, for which few annotated nucleic acid sequences exist, are cDNA subtraction libraries, differential display, exploiting PCR primers for known genes of other members of the family Cyprinidae and use of degenerate PCR primers designed from regions of moderate protein homology. Single or multiple genes noted as being differentially expressed in microarray analyses will then be used in separate studies to measure bioavailable stressors in the laboratory and field. These analyses will be accomplished by quantitative RT-PCR. Moving from analysis of single gene exposures to the global state of the transcriptome offers possibilities that those genes identified by DNA microarray analyses might be critical components of dynamic biological systems and networks, wherein chemical stressors exert toxic effects through various modes of action. Additionally, the ability to discriminate bioavailability of stressors in complex environmental mixtures, and correlation with adverse effects downstream from these early molecular events, presents challenging new ground to be broken in the area of risk assessment.