Endocrinological assessment of male reproductive toxicity was carried out in SD-Slc male rats treated with 5-FU (0, 20, 30 mg/kg/day) orally for 2-week or 4-week term. Serum hormone levels including GnRH, FSH, LH, prolactin, total and free testosterone, inhibin B, pro-alpha C, and activin A were determined as well as histopathological examination of the reproductive organs. The 5-FU treated groups showed histopathological changes in the testis such as degeneration of seminiferous epithelium. An obvious decrease in serum testosterone level was observed with a reduced organ weight of the seminal vesicle and prostate. However, no significant changes were noted in serum LH or FSH levels, nor in the morphological examination of the Leydig cells. Decreased serum levels were noted in activin A and prolactin. An increased serum level was noted in GnRH and pro-alpha C whose synthesis is regulated by FSH. Serum inhibin B levels showed a tendency toward decreasing with morphological change (vacuolation) in Sertoli cells. These results indicated that male reproductive toxicity induced by 5-FU would be augmented by decreased serum prolactin and testosterone levels as well as a decreased function of Sertoli cell, in addition to the direct cytotoxic effects on germ cells. It is suggested that these endocrinological changes related to male reproductive toxicities can be detected even in the 2-week-treated study.

No abstract

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.

The toxicity of Etretinate, a retinoid compound, on the male reproductive system was studied in male rats. The drug was administered for four weeks at the dose levels of 0 (control: Vehicle, Peanut oil), 5 and 25 mg/kg/day. The animals were then allowed to mate, and their male reproductive functions and organs were examined in detail. No significant changes due to toxicity were observed in male reproductive functions and organs in the 5 mg/kg/day group after the 4-week treatment. In contrast, males in the 25 mg/kg/day group showed drug-related changes in their reproductive performance (decrease of mating ability and fertility rate), testosterone blood level, sperm head counts, sperm viability and number in the caudal epididymis, organ weight and in the histopathology of their reproductive organs (atrophy of seminiferous tubules, necrosis of spermatocytes and spermatids, vacuolation of nuclei of spermatocytes and spermatids). Even though Etretinate belong to the retinoid group of compounds, the changes seen in the 25 mg/kg/day group were almost the same as those observed in Vitamin A-deficient animals. In conclusion, there is a correlation between changes due to toxicity observed for parameters of male fertility and for histopathological evaluation of the testis of rats that receiving high dose, treatment with Etretinate for 4 weeks.

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.