International Journal of Toxicology

The structure and function of the testis and epididymis are described, emphasizing the general similarities and specific differences between various species and humans. Current concepts of spermatogenesis are reviewed and the developmental stages of the germinal epithelium are discussed, as well as the complex hormonal interactions that take place. It is crucial to recognize that the efficiency of sperm production and the epididymal reserves in the human are considerably lower than those of conventional animal models. Therefore, the human male is more susceptible to a decline in fertility caused by a specific decrement in spermatogenesis than is an animal model.

Little is known about the factors controlling Leydig cell growth and differentiation. However, unique correlations exist between specific testicular compartments and the testosterone-secreting capacity of the testes. Selected experimental findings from three common laboratory animals—the rat, the hamster, and the guinea pig—are discussed.

The administration of 62.5 mg/kg/day of SDZ 200–110, a calcium channel blocker, for 2 years increased the incidence of Leydig cell tumors while decreasing pituitary tumors in Sprague-Dawley rats. Lower doses did not change the incidence of these tumors. No other endocrine tumors were seen in rats or mice of either sex. A single gavage dose of 62.5 mg/kg/day decreased serum testosterone levels by 90% 4 hr after dosing. In vitro testosterone production by Leydig cells from these animals was minimally decreased, which suggests that a direct inhibition of steroid synthesis was removed during cell isolation. Dietary administration of the drug for 10 weeks did not significantly alter levels of serum hormones or testicular luteinizing hormone (LH) and gonadotropin-releasing hormone (GnRH) receptors, although a significant elevation of testicular testosterone levels was seen. Increased serum levels of LH and follicle-stimulating hormone (FSH) were seen after 52 and 66 weeks, respectively, of dietary feeding of 62.5 mg/kg/day. The increase in serum LH was observed to week 104, while FSH levels returned to control levels by week 94. No effect on gonadotropin receptors was seen at the 6.25 mg/kg/day dosage. The age-related increase in serum prolactin was markedly reduced by 62.5 mg/kg/day of SDZ 200–110 in weeks 66 to 104 and to a lesser extent at the 6.25 mg/kg/day dosage. Testicular LH receptors were decreased by the high dose in animals sacrificed after 90–104 weeks. In conclusion, SDZ 200–110 increases the incidence of Leydig cell tumors by elevating levels of serum gonadotropins. The suggested mechanism for this increase in gonadotropins is a result of the effects of SDZ 200–110 on serum hormones and testicular LH receptors. The drug was judged not to pose a risk to humans since no change in gonadotropin levels was observed after chronic treatment.

This presentation will concentrate on the emerging field of flow cytometry. The first portion will be a review of the state-of-the-art applications of flow cytometry in the field of biomedical research; the second portion will describe the results of a preliminary study using a published technique that is useful in detecting cellular perturbations in germ cells. The model used in this study was the testis from the Sprague-Dawley rat. Adult rats received a single bolus of busulfan, and their testes were examined up to 56 days postadministration.

Groups of 25 male Sprague-Dawley rats were treated orally with losulazine at 0 (vehicle control), 4, 8, 16, or 32 mg/kg/day for 1 year. Daily clinical signs, weekly food consumption and body weight changes, and terminal hematologic and blood chemistry values were evaluated. Terminal urinalysis in 10 randomly selected rats from all groups and levels of serum luetinizing hormone, prolactin, and testosterone from control, low-, and high-dose groups were also evaluated. Fertility was determined in eight randomly selected rats from each group at 35–49 weeks. Reversibility of breeding performance was evaluated in 10 rats treated for 30 weeks and allowed to recover for 17 weeks. Selected organs were weighed and the testes and epididymides were microscopically evaluated in all rats that survived through the 1 year treatment period.

Rats treated with losulazine showed dosage-dependent ptosis, somnolence, fecal softening, and decreased food consumption with a corresponding retarded body weight gain. There were no biologically significant changes in hematologic, blood chemistry, or urinalysis values between treated and control rats. Relative spleen, heart, adrenal, and brain weights were increased in treated rats. There was a reversible dosage and time-dependent decreased fertility in rats treated with losulazine for 6–12 months. The incidence of testicular tubular atrophy/degeneration, usually confined to the subcapsular areas of the testes, and concentration of degenerate ge.minal cells in the epididymides, were increased in treated compared to vehicle control rats. Testicular lesions were not dosage related, were minimal to mild after 1 year of treatment, and were not attended by a decrease in relative testicular weights. Decreased fertility was not correlated with the apparently treatment-related testicular lesions. It could not be determined whether the minor testicular lesions seen in rats treated with losulazine were related to stressful conditions the rats were apparently under or to the effects of the drug on the hypothalamus-pituitary-gonadal axis or the sympathetic nervous system.

Many hormones are involved in regulation of the testis. Some of these act directly on the testis itself, while others act indirectly by influencing secretion or clearance of those that may act on the testis. Methods are not yet available for easily quantifying some of the hormones that affect testicular function, such as growth factors. Most hormones are secreted as pulses rather than in a constant, continuous manner. This necessitates appropriate sampling regimens to define the amplitude and frequency of the pulses. Factors such a stress, changes in environmental conditions, level of nutrition, and disease may alter the pattern and rate of secretion of hormones that regulate testicular function. All of these factors must be considered when designing experiments to determine how drugs affect the function of the testis.

The advantages of testicular sperm head counts are many, and the procedure should be encouraged. It is simple, quantitative, objective, and has excellent sensitivity. Effects can be measured for all stages of spermatogenesis, and indirect hormonal effects on spermatogenesis also can be detected. A large range for a dose-response curve exists, and there is a direct relationship to fertility in the animal. It is the only parameter that is really suitable for interspecies extrapolation, and in humans it is the parameter that can be applied to the quantitative assessment of reproductive risk.