Abstract.We investigated the long-term estrogenic influence of genistein on the male reproductive system in mice. Newborn ICR male mice were treated with genistein (10, 100, or 1000 , tg/mouse) for neonatal 5 days. As positive control, administration of diethylstilbestrol (0.5-50 pg/mouse) was carried out. In mice exposed to genistein,we examined weight of testes, sperm counts, sperm motility, and mRNA expression levels of estrogen receptor a (ERa) and androgen receptor (AR) at 4, 8 or 12 weeks after birth. Moreover, at 12 weeks of age, we evaluated protein level of ERa. In our conventional reproductive-toxicological study (weight of testes, sperm counts and sperm motility), neonatal transient exposure to genistein did not show adverse effects on the male reproductive system in 4, 8 or 12 week old mice. However, in mice treated with genistein mRNA expression levels of ERa and AR were reduced at 8 weeks. This reduction was recovered at 12 weeks in mice treated with a lower dose (10 ICg) of genistein but not in those with higher doses (100 pg and 1000 rig). In addition, ERa protein levels tended to decrease in 12 weeks of adulthood.Our results exhibited that the disruption of gene expression continued for long term such as 3 months after administration of genistein, even if no effect was found at conventional reproductive-toxicological level. We have shown that neonatal administration of weak estrogenic compound (genistein) affects male reproductive organs at molecular levels in adulthood.

We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays. Newborn male ICR mice were exposed to DES (50 microg/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis; 11 genes (AI035263, AU080565, AU080361, AU080678, AI131681, AU080631, AA986882, AI037066, AA986537, AI156816, and AI596237) were up-regulated and three genes (AI131656, AI118968, and AI117606) were down-regulated in DES-treated mouse testes. Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Real-time RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age. Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers.

We investigated whether neonatal exposure to diethylstilbestrol (DES) induces the alteration of mRNA expression in adult mouse epididymis, which plays an important role in sperm maturation. Using a cDNA subtraction method, we isolated 15 changed gene clones in neonatally DES-treated mouse epididymides, and we found a clone homologous with a disintegrin and metalloprotease (ADAM) 7 in the epididymis, as a suppressed gene, by means of neonatal DES treatment in 8-week-old mice. Indeed, it was confirmed by Northern blot analysis that the ADAM7 mRNA expression in the epididymis was at a lower level in neonatally DES-treated mice than in non-treated mice. Moreover, in situ hybridization analysis and real-time reverse transcription and polymerase chain reaction (real-time RT-PCR) revealed that ADAM7 expression was markedly reduced in the corpus region of the epididymis of DES-treated mice as compared with non-treated mice. Our results suggest that neonatal exposure to DES leads to the suppression of ADAM7 expression in the epididymis in the long term. ADAM7 gene expression might be a biological marker of fetal or neonatal exposure to estrogenic compounds, including endocrine disruptors.

Summary In utero or neonatal exposure to high levels of exogenous steroid hormones, such as the potent synthetic diethylstilbestrol (DES), incurs an increased risk of malfunctional male reproduction. In this study, we investigated whether neonatal exposure to DES induces the alteration of mRNA expression in adult mouse testis. Using a cDNA subtraction method, we isolated seven gene clones whose expression was changed in neonatally DES-treated mouse testis. Northern blot analysis revealed that five up-regulated genes (AF326230, AF356521, AK004975, AK006136 and BM237156) and two down-regulated genes (AK017044, AK017130) were predominantly expressed in testes of 8-week-old mice. Moreover, we confirmed that the expression of these seven genes was altered by neonatal DES-exposure using Northern blot analysis. Our results suggest that neonatal exposure to DES leads to the alteration of gene expression in the testis in the long term. These genes might be useful as biological markers of foetal or neonatal exposure to exogenous steroid hormones, such as DES.