Neonatal Exposure to Genistein Induces Estrogen Receptor (ER)α Expression and Multioocyte Follicles in the Maturing Mouse Ovary: Evidence for ERβ-Mediated and Nonestrogenic Actions

Abstract

Outbred CD-1 mice were treated neonatally on Days 1-5 with the phytoestrogen, genistein (1, 10, or 100 micro g per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor beta (ERbeta) predominated over ERalpha in controls and increased with age. Genistein treatment did not alter ERbeta expression, however, ERalpha expression was higher on Days 5 and 12. ERbeta was immunolocalized in granulosa cells, whereas ERalpha was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERalpha in granulosa cells. Genistein-treated ERbeta knockout mice showed a similar induction of ERalpha, which is seen in CD-1 mice, suggesting that ERbeta does not mediate this effect. Similar ERalpha induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERalpha expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERbeta must play a role, because mice lacking ERbeta showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.

Follicular assembly: mechanisms of action

“…Neonatal female mice treated with estradiol (E 2 ; , with synthetic estrogens such as diethylstilbestrol (DES; Iguchi et al 1990) or bisphenol-A (BPA; Suzuki et al 2002) or with the phytoestrogen genistein (Jefferson et al 2002), have more MOFs as adults, which are likely oocyte cysts that did not separate and became enclosed in follicles (Gougeon 1981. One model is that normally, high levels of E 2 in the fetal ovary keeps oocytes in cysts and that late in fetal development, E 2 levels drop resulting in cyst breakdown (Chen et al 2007).…”

“…To determine which ER genistein was signaling through, Jefferson et al (2002) exposed ER mutants to genistein as neonates and examined effects on the adult ovary. MOFs were still observed in ESR1 but not ESR2 mutant adult females, suggesting that these effects are mediated through ESR2 (Jefferson et al 2002). ESR2 mutant adult females have reduced fertility with fewer litters and pups per litter (Krege et al 1998).…”

Soy, phytoestrogens and their impact on reproductive health

“…These include alterations in ovarian development (increased percentage of multi-oocyte follicles (MOFs)), the timing of vaginal opening, estrous cyclicity, ovarian function, HPG axis, subfertility and an increased incidence of uterine adenocarcinoma (Chen et al, 2007;Delclos et al, 2001Delclos et al, , 2009Jefferson et al, 2006Jefferson et al, , 2002Jefferson et al, , 2005Kouki et al, 2003;Lewis et al, 2003;Nagao et al, 2001;Newbold et al, 2001;Nikaido et al, 2004;NTP, 2008). For example, in mice neonatal injection of genistein at doses of 0.5, 5 and 50 mg/kg/day on postnatal days 1-5 leads to prolonged estrous cycles with a dose-dependent and age-related increase in severity (Jefferson et al, 2002). These doses span the range of human exposure levels: a treatment of 50 mg/kg/day resulted in serum genistein levels of 6.8 lM, comparable to the circulating levels of 1.4-4.5 lM found in the serum of infants consuming soy-based formulas (Setchell et al, 1997).…”

“…While females exposed to low doses of genistein (0.5 and 5 mg/kg) showed signs of reduced fertility with age, mice treated with the highest dose of genistein (50 mg/kg) became pregnant at 2 months of age but were unable to support pregnancy and did not deliver any pups. Neonatal genistein treatment also results in a dose-dependent increase in the number of multi-oocyte follicles (MOFs) present in immature ovaries (Jefferson et al, 2006(Jefferson et al, , 2002. These effects appear to be mediated by ERb as MOFs are not observed in genistein-treated mice lacking ERb (Jefferson et al, 2002).…”

“…Neonatal genistein treatment also results in a dose-dependent increase in the number of multi-oocyte follicles (MOFs) present in immature ovaries (Jefferson et al, 2006(Jefferson et al, , 2002. These effects appear to be mediated by ERb as MOFs are not observed in genistein-treated mice lacking ERb (Jefferson et al, 2002). Interestingly, several other xenoestrogens including 17b-estradiol, DES and Bisphenol A have also been reported to affect ovarian development and cause MOFs in rodents (Iguchi et al, 1990;Suzuki et al, 2002).…”