-In the drug discovery process, effects to the human spermatogenesis must be fully evaluated before the first human trial. To estimate testicular toxicity, histopathological evaluation has been recommended in addition to the traditional mating procedure. However, it is laborious and time-consuming. Flow cytometric analysis (FCM) has also been applied to estimate testicular toxicity because of its speed, simplicity, and the objectivity of the data. Using cyclophosphamide (CP)-and ethinylestradiol (EE)-treated rat testis, we attempted to validate our dual-parameter, DNA ploidy and cell-size FCM, in a high-throughput toxicity study. Our results showed that CP damaged some spermatogonia and some early meiotic spermatocytes and EE caused severe decrease of spermatogenic cells except for spermatogonia as well as marked decrease of somatic cells, most probably Leydig cells. This is the first report discriminating between the changes of spermatogonia and that of somatic cells with FCM analysis. These results demonstrate that this method is a very useful and powerful tool to assess testicular toxicity, especially in high-throughput toxicological studies.

BackgroundThe in vitro micronucleus (MN) test is an important component of a genotoxicity test battery that evaluates chemicals. Although the standard method of manually scoring micronucleated (MNed) cells by microscope is a reliable and standard method, it is laborious and time-consuming. A high-throughput assay system for detecting MN cells automatically has long been desired in the fields of pharmaceutical development or environmental risk monitoring. Although the MN test per se cannot clarify whether the mode of MN induction is aneugenic or clastogenic, this clarification may well be made possible by combining the MN test with an evaluation of γH2AX, a sensitive marker of DNA double strand breaks (DSB). In the present study, we aimed to establish a high-content (HC) imaging assay that automatically detects micronuclei (MNi) and simultaneously measures γH2AX foci in human lymphoblastoid TK6 cells.ResultsTK6 cells were fixed on the bottom of each well in 96-well plates hypotonically, which spreads the cells thinly to detach MNi from the primary nuclei. Then, the number of MNi and immunocytochemically-stained γH2AX foci were measured using an imaging analyzer. The system correctly judged 4 non-genotoxins and 13 genotoxins, which included 9 clastogens and 4 aneugens representing various genotoxic mechanisms, such as DNA alkylation, cross-linking, topoisomerase inhibition, and microtubule disruption. Furthermore, all the clastogens induced both γH2AX foci and MNi, while the aneugens induced only MNi, not γH2AX foci; therefore, the HC imaging assay clearly discriminated the aneugens from the clastogens. Additionally, the test system could feasibly analyze cell cycle, to add information about a chemical’s mode of action.ConclusionsA HC imaging assay to detect γH2AX foci and MNi in TK6 cells was established, and the assay provided information on the aneugenic/clastogenic mode of action.Electronic supplementary materialThe online version of this article (10.1186/s41021-019-0117-8) contains supplementary material, which is available to authorized users.

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We investigated the application of flow cytometric analysis to evaluate the rat sperm viability and number in the male reproductive toxicity studies. Flow cytometric procedure has been developed to evaluate sperm number and viability that uses fluorescent dye (propidium iodide, PI) to distinguish between viable (negative staining) and dead (positive staining) sperm. Sperm samples were collected from the caudal epididymides of SD rats (13–21 weeks old). PI staining patterns/viabilities were compared among several ranges of sperm concentrations, and several kinds of sperm viability. Viabilities determined by flow cytometry (FC) were also compared with motility by direct microscopical observation in several kinds of sperm viability. No notable changes in the PI staining patterns/viabilities were observed in the range from 1 × 105 to 6 × 106 sperm/ml. Essentially similar results were obtained from both FC and microscopical analyses for three degrees of viability sperm: live sperm (general preparation as a control), weakly viable sperm (mixed by vortex mixer for 30 seconds), and dead sperm (treated with 90°C or Triton X‐100).Viabilities of normal rat samples were 95.0 ± 4.0% in FC and 93.7 ± 4.6% in microscopical observation, indicating good correlation in both analyses. Sperm numbers with FC analysis were approximately 0.8 × 106 to 1.8 × 106 sperm per mg indicating good correlation with those by microscopy.It was concluded that the present flow cytometric procedure was objective, rapid and reliable, and that it was one of the useful methods for measuring the number and evaluating the viability of sperm collected from the caudal epididymides of rats in the male reproductive toxicity studies.

Six-week old SD-Slc male rats were treated for 4 weeks with compounds known to induce toxicological changes in male reproductive organs (pyridoxine in saline, 500 mg/kg/day, i.p.) or sperm (trimethylphosphate in distilled water, 100 mg/kg/day, p.o.). Each sperm sample taken from the cauda epididymis was analyzed with flow cytometry for the evaluation of sperm viability and counts. Sperm motility and morphology by microscopical observation, and histopathological examination of reproductive organs were also performed for estimating the adverse effects of each compound on spermatogenesis and sperm. While a decrease in sperm motility was noted for the trimethylphosphate group, the low motile sperm was evaluated as being viable sperm, not moribund, with flow cytometry. In the pyridoxine group, microscopical observation revealed morphological changes of sperm and a decrease of motility. The present sperm analysis with flow cytometry also suggested morphological changes reflected by dot plot as well as decrease of sperm viability and counts. These results indicated that this procedure led to profound findings in the compound-treated animals, with evaluation as viable sperm, not moribund, in a low motile sperm sample, and suggesting morphological changes in the dot plot of flow cytometry.