David Edward Seyler

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: A consensus report☆

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Utilization of a short-term male reproductive toxicity study design to examine effects of α-chlorohydrin (3-chloro-1,2-propanediol)

alpha-Chlorohydrin (ACH) was administered to rats in a short-term male reproductive toxicity study to examine the usefulness of the method and to provide reference data with a substance that is known to elicit adverse effects on both sperm production and sperm quality within or following a 2-week treatment period. Adult male CD rats (10 per group) were administered ACH orally by gavage at doses of 0, 1, 5, or 25 mg/kg/day for 14 days. Males were killed on Test Day (TD) 15 or 29. A 2-week period without treatment was included to distinguish between testicular and posttesticular effects. At each time point, the reproductive system was evaluated by comparing testes weight, DNA ploidy distributions of testicular cell suspensions, testicular and epididymal histopathology, and epididymal sperm concentration, motility, morphology, and breakage. Beginning on TD 14, males to be killed on TD 29 were cohabited with untreated females (1:2). Females were killed on Gestation Day 13 and examined for pregnancy status. During the treatment period, minor depressions in body weight and relative food consumption occurred in rats administered 25 mg/kg ACH. Testicular and epididymal lesions also occurred at this dose level. DNA ploidy distributions determined by flow cytometry were predictive of testicular damage, with effects more pronounced on TD 29 than on TD 15. The preparation methods used selected for the most motile and vigorous population of epididymal sperm. Sperm motion was altered at the 5- and 25-mg/kg dose levels on TD 15. The percentage of motile sperm and the percentage of progressively motile sperm were markedly depressed at both the 5- and 25-mg/kg dose levels where antifertility effects occurred. Sperm velocities and amplitude of lateral head displacement were depressed at the 25-mg/kg dose level on both TD 15 and 29. Additionally, decreased epididymal sperm concentrations and increased breakage were recorded at this dose level. The findings in this study are consistent with the scientific literature for ACH and demonstrate posttesticular effects on epididymal sperm and delayed expression of testicular lesions. They also support the use of this methodology for an initial assessment of male reproductive effects.

Effect of growth hormone secretagogue LY444711 on IGF-1, growth hormone, and cortisol levels in beagle dogs after one and seven daily oral doses

Growth hormone (GH) release involves interaction of somatostatin and an endogenous GH secretagogue (GHS) on the hypothalamus. GH causes release of IGF‐1, which acts by negative feedback to restrain subsequent GH release. GH secretagogues produce increases in cortisol. In this study, we determined if compound LY444711 produces sustained elevation of GH and IGF‐1 in beagle dogs without sustained alteration of baseline cortisol secretions after one and seven daily doses. Adult male beagle dogs received oral doses of LY444711 at 1 mg/kg/day, or vehicle (10% hydroxypropyl beta‐cyclodextrin). Jugular vein blood was collected periodically after one and seven doses, and plasma levels of IGF‐1, GH, and cortisol were determined. LY444711 increased IGF‐1 levels by approximately 60% over controls after one and seven daily doses. IGF‐1 was elevated within 6 h of dosing on Day 1 and remained elevated 24 h postdose. GH levels (AUC) increased approximately 50‐fold above controls following a single dose of LY444711. With repeated dosing, GH levels rose to approximately 8‐fold over controls. Regardless of the reduction in GH AUC with repeat dosing, sufficient GH was produced to cause sustained IGF‐1 elevation after seven doses. LY444711 produced little or no effect on cortisol AUC level after one or seven doses. These data demonstrate that LY444711 functions as a GH secretagogue in dogs, with associated increases in IGF‐1 levels and an absence of meaningful increases in cortisol levels. Drug Dev. Res. 49:260–265, 2000.

Use of a rat ex-vivo testis culture method to assess toxicity of select known male reproductive toxicants

Due to the complex physiology of the testes, in vitro models have been largely unsuccessful at modeling testicular toxicity in vivo. We conducted a pilot study to evaluate the utility of the Durand ex vivo rat seminiferous tubule culture model [1-3] that supports spermatogenesis through meiosis II, including the formation of round spermatids. We used this system to evaluate the toxicity of four known testicular toxicants: 1,3-dinitrobenzene (DNB), 2-methoxyacetic acid (MAA), bisphenol A (BPA), and lindane over 21 days of culture. This organotypic culture system demonstrated the ability to successfully model in vivo testicular toxicity (Sertoli cell toxicity and disruption of meiosis) for all four compounds. These findings support the application of this system to study molecules and evaluate mechanisms of testicular toxicity.

Developmental Toxicity of Amesergide Administered by Gavage to CD Rats and New Zealand White Rabbits

Amesergide is a selective serotonin 5-HTIC/2 receptor antagonist being developed for the treatment of depression. The potential developmental toxicity of amesergide was evaluated in CD rats and New Zealand white rabbits. Pregnant rats and rabbits were dosed once daily by gavage on Gestation Days 6-17 and 6-18, respectively. Doses for rats were 0, 3, 10, and 30 mg/kg; doses for rabbits and 0, 0.2, 2, and 15 mg/kg. Cesarean sections were performed on rats and rabbits on Gestation Days 20 and 28, respectively. In rats, maternal effects expressed as depression of body weight gain and food consumption were observed at the 30 mg/kg dose level. Fetal viability and morphology were not affected at any dose level. Fetal weight was depressed at the 30 mg/kg dose level. The no-observed-effect level (NOEL) in the rat was 10 mg/kg. In rabbits, maternal effects expressed as a decrease in food consumption occurred at the 2 and 15 mg/kg dose levels; weight gain was depressed at 15 mg/kg. Fetal viability, weight, and morphology were not affected at any dose level. The NOELs for maternal and developmental effects in the rabbit were 0.2 and 15 mg/kg, respectively.

Effects of the serotonin antagonist amesergide on reproduction in female rats

Amesergide, a serotonin (5-HT2) antagonist intended to treat depression, was administered orally to female CD rats (20/group) at doses of 0, 3, 10, or 30 mg/kg to evaluate effects on mating, fertility, litter size, live birth index (100 x total liveborn progeny/litter size), progeny survival, and weight gain of each litter. The treatment period extended from two weeks prior to mating through postpartum day 21 to cover possible effects of estrous cycle, mating, gestation, and postpartum events. Twenty additional female rats were given 30 mg/kg through gestation day 18, after which they received the acacia vehicle (recovery group). All females were allowed to deliver naturally and rear their progeny. On postpartum day 8, progeny in the control, 30 mg/kg and 30 mg/kg recovery groups were removed from dams for 4 h. Progeny were weighed as litters, returned to the dams for a 1-h nursing period, and then weighed again to provide an indication of milk intake. Mating and fertility, using the present study design, were not affected by treatment with amesergide. No effects were observed on litter size, live birth index, or progeny survival. In contrast, treatment with amesergide throughout gestation and lactation produced a significant dose-related depression in progeny body weight gains. However, when treatment was discontinued after day 18 of gestation (30 mg/kg recovery group), progeny body weight gains did not differ from those of the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

A 40-week male fertility study of the anticancer agent sulofenur (LY186641) administered orally to rats.

Sulofenur was evaluated for fertility effects on rats. Five-week old male rats (20/group) received 0, 5, 30, or 60 mg Sulofenur/kg/day for 14 weeks. Fertility was evaluated five times. Treated males were mated with untreated females at 10 weeks. Half the males from each group were necropsied after the 14-week treatment period and the remainder were mated four additional times during a 26-week posttreatment period. Following each mating, females were killed on gestation-day 20 and examined for evidence of pregnancy. Six weeks after mating trial 5, the remaining males were necropsied. Testes and epididymides were collected, weighed, and examined microscopically. All rats mated in each mating trial, and all control and low-dose animals were fertile. A significant reduction in fertility occurred in the middle- and high-dose males. This was consistent with testicular hypospermatogenesis in these groups. Fertility recovered in the high-dose group following cessation of treatment, but remained reduced in the middle-dose group. Preimplantation and postimplantation loss in mating trial 1 were higher in the middle- and high-dose groups. Abnormal fetuses were present in the high-dose group in mating trial 3, but not in mating trials 4 or 5. In conclusion, male rats given doses of 30 and 60 mg/kg/day of Sulofenur showed hypospermatogenesis and decreased fertility. Spermatogenesis and fertility recovered in the high-dose group. A dose of 5 mg/kg/day did not produce any effect on testes or fertility.