Effects of genistein in the maternal diet on reproductive development and spatial learning in male rats

Abstract

Endocrine disruptors, chemicals that disturb the actions of endogenous hormones, have been implicated in birth defects associated with hormone-dependent development. Phytoestrogens are a class of endocrine disruptors found in plants. In the current study we examined the effects of exposure at various perinatal time periods to genistein, a soy phytoestrogen, on reproductive development and learning in male rats. Dams were fed genistein-containing (5 mg/kg feed) food during both gestation and lactation, during gestation only, during lactation only, or during neither period. Measures of reproductive development and body mass were taken in the male offspring during postnatal development, and learning and memory performance was assessed in adulthood. Genistein exposure via the maternal diet decreased body mass in the male offspring of dams fed genistein during both gestation and lactation, during lactation only, but not during gestation only. Genistein decreased anogenital distance when exposure was during both gestation and lactation, but there was no effect when exposure was limited to one of these time periods. Similarly, spatial learning in the Morris water maze was impaired in male rats exposed to genistein during both gestation and lactation, but not in rats exposed during only one of these time periods. There was no effect of genistein on cued or contextual fear conditioning. In summary, the data indicate that exposure to genistein through the maternal diet significantly impacts growth in male offspring if exposure is during lactation. The effects of genistein on reproductive development and spatial learning required exposure throughout the pre- and postnatal periods.

Fig. 1

Water maze swim strategies. Representative examples of the various swim strategies used by the rats are shown as tracings of the swim path. In each tracing the large circle represents the edge of the pool and the small double circle represents the location of the hidden platform. Rats tended to use thigmotaxis as their initial strategy. As training progressed, rats tended to shift their strategy to a non-spatially biased swim path such as circling or scanning and then to a spatially biased path with the ultimate path being a direct swim to the platform.

Fig. 2

Maternal behavior of control and genistein-treated dams. (A) Dams that ate genistein throughout gestation and lactation (G-G) or only during gestation were observed to nurse significantly more than control dams (C-C) as indicated by the asterisk (p < 0.05, Fisher LSD) (B) Pup licking did not differ between the groups. (C) Dams' contact with the nest did not differ between the groups. Observations were performed everyday between PND 3 and 21. All data are pooled by blocks of postnatal days and expressed as the mean ± SEM.

Fig. 3

Effect of pre- and postnatal genistein on anogenital distance. Exposure to genistein (5 mg/kg of food) via the maternal diet during gestation and lactation (G-G) significantly reduced anogenital distance compared to exposure to phytoestrogen-free diet during gestation and lactation (C-C). Asterisks indicate p < 0.001 as determined by the Tukey post hoc test. Data are presented as the grand mean of litter means ± SEM; litter n's were 8-9 for each group.

Fig. 4

Effect of pre- and postnatal genistein on postnatal growth. Exposure to genistein (5 mg/kg of food) via the maternal diet during gestation and lactation (G-G) and during lactation only (C-G) significantly reduced body mass on postnatal day 21 compared to exposure to phytoestrogen-free diet during gestation and lactation (C-C). Asterisks indicate p ≤ 0.01 as determined by the Tukey post hoc test. Data are presented as the grand mean of litter means ± SEM; litter n's were 8-9 for each group.

Fig. 5

Water maze performance in the hidden (A) and visible (B) platform tasks. (A) Exposure to genistein during gestation and lactation (G-G) significantly increased the latency to find the platform on day 3 of training compared to the unexposed group (C-C). Asterisk indicates p < 0.001 as determined by the Tukey post hoc test. (B) There was no effect of genistein exposure on the latency to find the visible platform. Data are presented as the mean ± SEM, and n's were C-C = 13, G-G = 7, G-C = 6, C-G = 11. Probe trials were performed on days 7 and 10.

Fig. 6

Swim path analysis of hidden platform acquisition trials. (A) Exposure to genistein during both gestation and lactation (G-G) significantly increased thigmotaxis on day 3 of training relative to controls. Asterisk indicates p < 0.05 by the Tukey post hoc test. Data are the mean ± SEM. (B) Swim path identification showed that rats exposed to genistein through both gestation and lactation (G-G) had their last day of relying on thigmotactic swimming significantly later than C-C rats. Asterisk indicates p = 0.007 (Mann-Whitney test). The bold horizontal line in each box indicates the median. Boxes extend to the 25^th and 75 percentiles. Whisker caps extend to the 10^th and 90^th percentiles. The data point is a subject that was below the 10th percentile. (C-D) Swim paths were identified and categorized for C-C (panel C) and G-G (panel D) rats as described in the Methods. The stacked bars in the graph show the percentage of rats that used thigmotaxis (black), non-spatial (white), or spatial (gray) strategies on the first trial of each training day. Note that on days 3 and 4 thigmotaxis is disappearing as a strategy to a greater extent in the C-C rats.

Fig. 7

Water maze probe trial performance. All groups spent significantly more time in the quadrant that had contained the platform (target) during the acquisition of the hidden platform task than in the adjacent left, right, or opposite quadrants. (A) control group (C-C). (B) gestation and lactation group (G-G). (C) gestation only group (G-C). (D) lactation only group (C-G). Asterisks indicate p < 0.001. Data are presented as the mean ± SEM.

Fig. 8

Cued and contextual fear conditioning. Pre- and postnatal genistein exposure through the maternal diet had no effect on freezing behavior during training (A), testing of conditioning to the training context (B), or testing of conditioning of the cue (C). Data are presented as the mean ± SEM; n's were C-C = 13, G-G = 7, G-C = 6, C-G = 10.