Return to fertility, toxicology, and transgenerational impact of treatment with WIN 18,446, a potential male contraceptive, in mice

Abstract

Objectives:

We examined the return to fertility and transgenerational impact of treatment with WIN 18,446, an experimental male contraceptive, in mice.

Study design:

We paired male mice treated with WIN 18,446 for 4 weeks to suppress spermatogenesis, followed by a 9-week recovery, and mated them with normal females to assess fertility. F1 generation mice were subsequently mated to ascertain any transgenerational impact of treatment on fertility. Testes were examined histologically.

Results:

WIN 18,446–treated mice and their progeny produced normally sized litters (6.5 pups per litter after treatment and 7.3 pups per litter from the progeny). However, testes histology revealed rare residual intratesticular foci of mineralization after treatment.

Conclusions:

Fertility normalizes after WIN 18,446 treatment, and progeny also have normal fertility.

1. Introduction

Local biosynthesis of the vitamin A metabolite retinoic acid (RA) plays essential roles in gametogenesis [1,2]. RA is synthesized in animals by the aldehyde dehydrogenase 1A [ALDH1A1] enzymes, 1A2 and 1A3. Testes-specific deletion of the genes encoding Aldh1a1 and 1a2 in mice inhibits spermatogonial differentiation, causing male infertility [3,4]. Similarly, inhibition of RA biosynthesis with oral administration of the ALDH1A inhibitor WIN 18,446 inhibits spermatogenesis in many species, including man [5–7]. Unfortunately, side effects from the inhibition of other ALDH isozymes by WIN 18,446 prevented its further development as a male contraceptive. Nevertheless, ALDH1A-specific inhibitors may be useful as male contraceptives. However, the long-term effects of inhibition of RA biosynthesis on return to male fertility—both in treated males and their offspring—have not been directly studied. Therefore, in this study, we sought to ascertain the return to fertility of WIN 18,446–treated mice after recovery of spermatogenesis in a mating experiment. In addition, to determine any transgenerational effect on fertility of WIN 18,446 treatment, we examined the fertility of mice fathered by WIN 18,446–treated animals. We hypothesized that the fertility of WIN 18,446–treated animals and their progeny would be normal compared with untreated control animals.

2. Materials and methods

Six-week-old male C57BL/6J mice (n = 25) were purchased from the Jackson Laboratory and acclimated in our facility as described previously [8]. Mice were treated with WIN 18,446 (2 mg/g diet) for 5 weeks, which suppresses spermatogenesis and fertility [4]. At the end of the drug treatment, five mice were euthanized, and the testes were weighed and processed for histologic analysis. In addition, liver was analyzed as we previously showed that WIN 18,446 treatment was associated with hepatic lipidosis [4]. After 4 weeks of treatment with WIN 18,446, the remaining 20 mice were switched back to a regular chow diet (5053; LabDiet). Nine weeks following the drug cessation, five additional mice were euthanized, and the testes and liver were examined. Ten of the 15 remaining posttreatment mice were mated with females in a 1:1 ratio to determine posttreatment fertility. Once pregnancies occurred, litter size was recorded. At the end of the mating period, liver and testes samples were collected. The five mice that were not used for mating were also euthanized to collect testes and liver. Progeny (both males and females) of the WIN 18,446–treated male mice were mated with each other to determine their fertility. For this study, four progenies (two males and two females) per sire were randomly selected and mated (1:1) until they produced a litter. Once they produced a litter, the number of pups was recorded, and males were euthanized, and the testes were collected for study. A schematic representation of the studies is provided in Supplementary Figure 1. Age-matched C57BL/6J mice from our colony at 13, 22, and 30 to 40 weeks of age were used to determine the testes weight of untreated mice at different ages and to rule out age-related histologic changes in 30- to 40-week-old mice. All tissues were processed and stained with hematoxylin and eosin. Tissue samples were examined and scored by a board-certified veterinary pathologist in a blinded fashion using standard scoring methods (shown in Supplementary Table 1).

3. Results

3.1. Mice and their offspring are fertile after recovery from WIN 18,446 treatment

After 4 weeks of treatment with WIN 18,446 and 9 weeks of recovery, nine of 10 treated males produced progeny when mated with a female. Each breeder produced at least one litter, and several produced three litters during 15 weeks of cohabitation. The litters ranged in size from three to nine pups per litter (average of 6.5 pups per litter), well within the normal range for this strain of mice [9]. To determine if the WIN 18,446 treatment influenced the fertility of progeny, we paired 14 male and 14 female offspring and monitored litter production. All 14 pairs produced a litter within 5 weeks of pairing. Litter size ranged between five and nine pups per litter, with an average of 7.3 pups per litter.

3.2. Testes and liver histology

As observed previously, treatment with WIN 18,446 for 4 weeks significantly reduced testes weight (Fig. 1, Supplementary Table 2) and suppressed spermatogenesis (Fig. 2A) [7]. After 9 weeks of recovery, testes’ weight returned to the normal range (∼100 mg per testis) [10], although testes’ weight was significantly lower than age-matched (22 weeks old) C57BL/6 males in our colony. In contrast, testes’ weight of the F1 progeny males measured at 12 weeks of age was similar to age-matched males in our colony. After 4 weeks of WIN 18,446 treatment, histologic analysis confirmed that WIN 18,446 treatment resulted in testes with only Sertoli cells and spermatogonia in most tubules, and rare mineralization involving one to two seminiferous tubules was observed. After 9 weeks of recovery, histology was markedly improved, and most tubules displayed complete spermatogenesis. Less than 5% of tubules (one to three tubules per testis) had mineralization in three of the five mice from this group (Fig. 2B), which persisted until the end of the study in recovered animals (Fig. 2C). For comparison, testes from untreated colony mice that were aged 30 to 40 weeks were also analyzed. These older mice also had rare degenerative/atrophic tubules (Fig. 2D), although they were not mineralized in the same way as the post-WIN 18,446–treated animals. As expected, we also observed hepatic cytoplasmic vacuolation (lipidosis) in mice treated with WIN 18,446 (Fig. 2E). However, after 9 weeks of drug cessation, the lipidosis was completely reversed. We did not observe any abnormalities in testes or liver of F1 offspring (Fig. 2E and J).

Fig. 1.

Testes weight. A pair of testes were weighed at indicated time points from experimental animals and age-matched colony males (9-wk recovery vs 22 week old; 25-wk end point vs 30–40 wk; F1 progeny and 4 wk tx vs 13 wo). Horizontal bars represent mean ± SD. **** < 0.0001; ** < 0.001. NS = not significant; tx = treatment, wo = week old (colony males).

Fig. 2.

Testes (top row): After 4 wk of treatment with WIN 18,446, diffuse tubular degeneration/atrophy is observed with depletion of spermatocytes (panel A). This is markedly improved in mice after the 9-wk recovery (panel B), although rare tubular degeneration/atrophy and mineralization are observed (arrows) in mice after 9-wk recovery and at the study end point (panel C). In the 30- to 40-wk-old control colony mice, rare tubular degeneration and atrophic tubules are observed in some mice (panel D, arrow). Testes from progeny mice are histologically normal (panel E). Liver (bottom row): After 4 wk of treatment with WIN 18,446, there is mild, multifocal, macrovesicular, and lesser microvesicular cytoplasmic hepatocyte vacuolation consistent with lipidosis (panel F, arrow). This is not observed in the 9-wk recovery (panel G), study end (panel H), control (panel I), or progeny (panel J) groups. Hematoxylin and eosin. Liver images are at 100× original magnification, and testes images are at 200× original magnification. tx = treatment, wo = week old

4. Discussion

WIN 18,446 treatment for 4 weeks suppressed spermatogenesis and induced mild hepatic lipidosis. After 9 weeks of drug cessation, the animals demonstrated a return to normal spermatogenesis and fertility, and the hepatic steatosis resolved. Importantly, there was no indication of a transgenerational impact of WIN 18,446 on either male or female offspring of WIN 18,446–treated animals. However, we observed mineralization in rare seminiferous tubules in the drug-treated group (one to three affected tubules per testis examined histologically) that was not seen in controls and that has not previously been reported with WIN 18,446 treatment. At this time, we do not know why occasional mineralization of the testes occurs in some mice treated with and recovered from WIN 18,446 and whether these findings are specific to WIN 18,446 treatment, ALDH1A inhibition in general or all forms of male contraception. Mineralization in mouse testis has been reported as a sequela to sperm stasis within the seminiferous tubules and may occur as an age-related lesion [11]. It will be important to determine if the enzymes that are required for RA biosynthesis and sperm production can be inhibited by male contraceptives without causing these abnormalities. Therefore, careful safety studies with longer periods of exposure in multiple species will be needed for male contraceptives such as WIN 18,446 that target RA biosynthesis or the retinoic signaling pathway to ensure that no testicular injury occurs with protracted exposure.