Genistein impairs early testosterone production in fetal mouse testis via estrogen receptor alpha

Toxicology in Vitro 25 (2011) 1542–1547

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Genistein impairs early testosterone production in fetal mouse testis via estrogen receptor alpha Abdelali Lehraiki a,b,c, Cathie Chamaillard a,b,c, Andrée Krust d, René Habert a,b,c, Christine Levacher a,b,c,⇑ a

Laboratory of Gonad Differentiation and Radiobiology, Stem Cells and Radiation Service, Institute of Cellular and Molecular Radiation Biology, Life Sciences Division, Commissariat à l’Energie Atomique, B.P. 6, 92265 Fontenay-aux-Roses, France b Université Paris Diderot-Paris 7, B.P. 6, 92265 Fontenay-aux-Roses, France c Unité 967, INSERM, B.P. 6, 92265 Fontenay-aux-Roses, France d Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS, INSERM, UDS, Collège de France), B.P. 10142, 67404 Illkirch-Strasbourg, France

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Article history: Received 28 October 2010 Accepted 16 May 2011 Available online 23 May 2011 Keywords: Phytoestrogen Leydig cell Steroidogenesis Gonocyte Estrogen Development

a b s t r a c t The widespread consumption of soy-based products raises the issue of the reproductive toxicity of phytoestrogens. Indeed, it is well known that genistein, an isoflavone found in soybeans and soy products, mimics the actions of estrogens and that the fetal testis is responsive to estrogens. Therefore we investigated whether genistein could have deleterious effects on fetal testis. Using organ cultures of fetal testes from wild type and ERa or ERb knock-out mice we show that genistein inhibits testosterone secretion by fetal Leydig cells during early fetal development (E12.5), within the ‘‘masculinization programming window’’. This effect occurs through an ERa-dependent mechanism and starting at 10 nM genistein, a concentration which is compatible with human consumption. No effect of genistein on the number of gonocytes was detected at any of the studied developmental stages. These results suggest that fetal exposure to phytoestrogens can affect the development and function of the male reproductive system. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Estrogens play a role also in the development and functions of the male reproductive system (Carreau et al., 2008; Delbes et al., 2006; Scott et al., 2009; Sikka and Wang, 2008; Toppari, 2008), as attested by the phenotype of male mice in which estrogen receptors (ER) (Eddy et al., 1996; Krege et al., 1998; Dupont et al., 2000) or the cyp 19 aromatase (Robertson et al., 1999) had been deleted and by the poor quality of semen from male patients with mutations in the ERa gene and from individuals suffering from aromatase deficiency (Morishima et al., 1995). The male reproductive tract and the testis itself are responsive to estrogens from fetal life throughout adulthood since they express the estrogen receptors ERa and ERb and estrogen-related receptors from early stages of embryonic development (Fisher et al., 1997; Mitsunaga et al., 2004; Nielsen et al., 2000). Moreover, testis development is physiologically modulated by estrogen. Indeed, ERb is expressed in germ cells during late fetal life (Jefferson et al., 2000) and ERb inactivation leads to an increase in the number of

⇑ Corresponding author. Address: Unité Mixte ‘‘Cellules Souches et Radiations’’, INSERM U967/CEA/Université Paris Diderot, LDRG/SCSR/IRCM/DSV, Centre CEA, B.P. 6, 18 Route du Panorama, Bat. 05A RdC, 92265 Fontenay-Aux-Roses, France. Tel.: +33 6 63 38 62 03; fax: +33 1 46 54 99 06. E-mail address: [email protected] (C. Levacher). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.05.017

gonocytes in neonates (Delbes et al., 2004). On the other hand, ERa is expressed in Leydig cells (Nielsen et al., 2000) and ERa inactivation (Delbes et al., 2005) enhances testosterone production throughout fetal life. Fetal testis is also particularly sensitive to endocrine disruptors (EDs). In utero or neonatal exposure to exogenous estrogens (diethylstilbestrol (DES), ethinylestradiol, bisphenol A, etc.) leads to reproductive anomalies in adulthood (Sharpe and Irvine, 2004; Sharpe et al., 2003; Skakkebaek et al., 2001; Toppari et al., 1996). Similarly, in vitro exposure of rodent fetal testis to DES or estradiol decreases testosterone synthesis (Delbes et al., 2004, 2005; Lassurguere et al., 2003) and reduces the number of gonocytes (Delbes et al., 2007; Lassurguere et al., 2003). Phytoestrogens are non-steroidal compounds that can bind to and activate both ERa and ERb due to their ability to mimic the conformational structure of estradiol (Kuiper et al., 1997, 1998; Le Maire et al., 2010) and are therefore classified as EDs. Genistein and daidzein are the predominant isoflavones in soy-based products. In humans who have a soy-free diet, the plasma concentration of isoflavones is usually in the nanomolar range (640 nM) (Morton et al., 1994; Van Erp-Baart et al., 2003) and genistein and daidzein concentration in the amniotic fluid before twenty weeks of gestation is about 5 nM (Foster et al., 2002). Conversely, in Asian populations with high consumption of soy-based products, blood levels of isoflavones range from 100 to 500 nM (Setchell, 1998). Moreover, in adults, the serum level of genistein can

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reach 10 lM shortly after intake of phytoestrogen-rich food (Adlercreutz et al., 1993; Busby et al., 2002; King and Bursill, 1998; Watanabe et al., 1998; Xu et al., 1994). In recent years, consumption of soy products by Western populations has been increasing because of the beneficial effects that have been attributed to phytoestrogens, such as prevention of cardiovascular diseases and hormone-dependent breast cancer, and their capacity to lower cholesterol (Cederroth and Nef, 2009; Park et al., 2005). Since such higher concentrations of genistein can have estrogenic effects in vivo and in vitro (Penza et al., 2007; Akingbemi et al., 2007), they might also lead to reproductive toxicity. The effects of phytoestrogens on the male reproductive function are controversial. Some studies did not observe detrimental effects on male reproductive physiology after exposure to isoflavones (Delclos et al., 2001; Faqi et al., 2004; Fielden et al., 2003), while others have shown that high levels of phytoestrogens, due to lifetime exposure or during a critical period of development, have detrimental effects on fertility and reproductive functions. Indeed, consumption of soy-based food modulates serum androgen levels in rodents and non-human primates (Sharpe et al., 2002; Tan et al., 2006; Wisniewski et al., 2003) and may affect male reproductive functions (Cederroth et al., 2010; Eustache et al., 2009). Moreover, subcutaneous administration of 4 mg/kg/day genistein to neonatal rats stimulated germ cell development in comparison to control animals (Atanassova et al., 2000). Nutritional isoflavones can bind to ERa and ERb and have both ER-agonist and ER-antagonist effects in a tissue-specific and promoter-specific manner (Schultz et al., 2005). Genistein is known to have estrogenic effects at low doses by binding to ERa and ERb (Le Maire et al., 2010), although at higher doses it also interacts with other nuclear receptors such as PPARs (Dang et al., 2003) and affects the activation of protein kinases, apoptosis and cell proliferation (Huang et al., 2005; Yu et al., 2004). Besides its estrogenic activity, genistein has also been reported to act through other mechanisms, for instance, as a tyrosine kinase inhibitor or as an anti-oxidant (Akiyama et al., 1987; Vedavanam et al., 1999). Therefore many aspects of the effects of genistein on the development of the fetal testis need to be clarified. In this study we used an organ culture system (Livera et al., 2006), in which the testicular architecture is conserved and development is similar to what observed in vivo, to address four questions: (1) Do concentrations of genistein compatible with human consumption have an effect on fetal testis? (2) Since phytoestrogens might affect both germ and somatic testicular cells, which cells are more vulnerable to genistein effects? (3) What are the pathways involved, since it is not clear whether the effects of genistein are due to the activation of ER-mediated pathways? and (4) Is there a sensitive period in fetal life during which genistein specifically alters testosterone production and germ cell development, as previously shown for estradiol and DES (Delbes et al., 2007; Lassurguere et al., 2003), other ED-like phthalates (Lehraiki et al., 2009) and other modulators of testicular development, like retinoic acid (Livera et al., 2000, 2001, 2004)?

2. Materials and methods 2.1. Chemicals and solutions The culture medium was phenol red-free Dulbecco’s Modified Eagle Medium/Ham F12 (1:1) (Invitrogen, Carlsbad, CA) supplemented with 80 lg/ml gentamicin (Invitrogen). Ovine luteinizing hormone (oLH; NIH.LH S19; 1.01 IU/mg) was a gift from Dr. A.F. Parlow (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Genistein was from Sigma–Aldrich (St Louis, MO).

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2.2. Animals C57Bl/6 and transgenic mice were housed under controlled photoperiod (lights on between 08:00 and 20:00) with ad libitum access to tap water and a soy- and alfalfa-free breeding diet (Global diet 2019, Harlan Teklad, Indianapolis, IN). ERa (ERa /) and ERb (ERb /) knock-out mice were generated by Dupont et al. (2000) and generously provided by Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France). Transgenic animals were genotyped as described previously (Delbes et al., 2004, 2005). Males were caged with females overnight and the day after was counted as embryonic day 0.5 (E0.5). Pregnant mice were killed by cervical dislocation at E12.5 or E18.5 and embryos were quickly removed from the uterus. Fetuses were dissected under a binocular microscope, their sex determined on the basis of recognizable gonad morphology and testes were collected from male fetuses. Natural birth occurred at E19.5 and was counted as post-natal day 0 (PN0). PN1 male neonates were killed by decapitation and their testes immediately removed. All animal studies were conducted in accordance with the guidelines for the care and use of laboratory animals of the French Ministry of Agriculture. 2.3. Organ culture and treatment Organ cultures were performed as previously described (Livera et al., 2006). Briefly, intact E12.5 testes and their mesonephric tissue were placed on 10-mm-diameter Millicell CM filters (Millipore, Billerica, MA) (pore size: 0.45 lm). Testes from E18.5 and PN1 mice were isolated and cut into eight and twelve pieces, respectively, and all the pieces from the same testis were placed on a single Millicell filter. The filters with the testes were then floated on 0.4 ml of culture medium in tissue culture dishes and incubated at 37 °C, in a humidified atmosphere containing 95% air and 5% CO2. The medium was changed every 24 h and culture was continued for 3 days. The effect of genistein was estimated by culturing one testis in the presence of genistein and the contra-lateral testis in control medium. In some cases testes were cultured in the presence of 100 ng/ ml oLH. At the end of culture, testes were fixed for 1 h at room temperature in Bouin’s fluid, embedded in paraffin and cut into 5-lm sections. For RNA analysis, testes were immediately dry frozen with liquid nitrogen and stored at 80 °C. Spent medium was kept at 20 °C for testosterone radioimmunoassay. Data were obtained from at least three independently repeated cultures of testes from different litters. 2.4. Testicular cell counting and immunohistochemistry Serial sections were mounted on slides, deparaffinized and rehydrated. They were immunostained using a standard, previously described procedure (Delbes et al., 2007). For detection of the Mouse Vasa Homolog (MVH) protein, sections were microwaved at 750 W for 5 min and at 450 W for 3 min in 10 mM citrate buffer solution (pH 6.0). For all immunohistological procedures, slides were incubated in 0.3% H2O2 for 15 min and in 5% bovine serum albumin in phosphate-buffered saline for 30 min to block non-specific antigens and were then incubated with the rabbit polyclonal anti-3bHSD (anti-3beta Hydroxysteroid Dehydrogenase) antibody (1/5000; provided by Prof. A. Payne, Stanford University Medical Center) or the rabbit anti-MVH antibody (1/500; Abcam, Cambridge, UK) at 4 °C overnight. The primary antibody was detected by incubation at room temperature with an appropriate biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 30 min followed by incubation with the avidin–biotin peroxidase complex (Vector Laboratories) for 30 min. DAB (3,30 -diaminobenzidine, Vector Laboratories) was used as

2.7. Statistical analysis All values are expressed as means ± SEM. The statistical significance of the difference between the mean values of treated and untreated testis from the same fetus was evaluated using the paired Student’s t-test. The unpaired Student’s t-test was used to evaluate the difference between the mean values of two different genotypes. 3. Results 3.1. Effects of genistein on Leydig cells In order to investigate the effects of genistein on embryonic and neonatal testes, testes in organ culture were exposed to different concentrations of genistein for 3 days and then testosterone secretion in the medium, testis morphology and number of germ/somatic cells were determined. Testosterone secretion by E12.5 testes during 3 days of culture was reduced following incubation with genistein in a dose- and time-dependent manner (Figs. 1 and 4). Specifically, 1010 M genistein did not have any effect, while 108 and 106 M genistein inhibited testosterone secretion in dose-dependent way, although at 105 M the inhibition was weaker (Fig. 1). Compared with controls, the number of Leydig cells was not modified after exposure to 106 M genistein (Fig. 2A). Moreover,

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Fig. 1. Genistein inhibits testosterone secretion by E12.5 fetal testes. Testes were cultured in the presence of various concentrations of genistein for 3 days. Testosterone secretion is expressed as the percentage of the control values. Values are the mean ± SEM from 6 to 10 cultures. ⁄p < 0.05, ⁄⁄p < 0.01 versus the corresponding control value (paired Student’s t-test).

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At the end of the culture period, total RNA was extracted from a pool of three testes using the RNeasy Plus mini-Kit (Qiagen, Courtaboeuf, France) and 500 ng of RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Courtaboeuf, France) according to the manufacturer’s instructions. To determine the levels of mRNA coding for proteins implicated in the steroidogeneic pathway (steroidogenic acute regulatory protein (StAR), p450 side chain cleavage (p450scc), 17alpha-hydroxylase-C17-20 lyase (p450C17) and 3beta hydroxysteroid dehydrogenase (3b-HSD), real-time quantitative PCR (qPCR) amplification was performed on an ABI PRISM 7000 Sequence Detector System using the TaqMan PCR Master Mix (Applied Biosystems). Some primers and probes were designed by Applied Biosystems (Courtaboeuf, France) (sequences not provided, P450c17, Mm00484040-m1; P450scc, Mm00490735-m1; StAR, Mm00441558-m1; b-actin, Mm00607939-s1). Primers for 3bHSD were: F: 50 -CTCAGTTCTTAGGCTTCAGCAATT-30 , R: 50 -AAGGCAGGATATGATTTA-30 and the internal probe 50 -TTTCACTTAGAAC TTAGT-30 . Results were analyzed using the delta-delta Ct method and normalized to b-actin expression. For each treatment, the mRNA levels were determined in samples from three different litters and at least from six to nine testes.

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Testosterone secretion into the medium was determined in duplicate by direct radioimmunoassay, without extraction as previously described (Habert and Picon, 1984).

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the chromogen and hematoxylin as nuclear counterstain. Negative controls were done by omitting the primary antibody. Germ cells and Leydig cells were identified by immunohistochemical detection of MVH and 3bHSD, respectively. The counting was done as previously described (Delbes et al., 2004). Counting was performed blind to the testis culture conditions using the Histolab analysis software (Microvision Instruments, Evry, France).

Testosterone secretion (% of control)

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Number of Leydig cells / testis (x106)

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Fig. 2. Genistein reduces the steroidogenic activity of E12.5 testes without modifying the number of Leydig cells. (A) Number of Leydig cells in E12.5 testes after 3 days of culture in the absence (white bars) or presence (black bars) of 106 M genistein. Leydig cells were identified by immunohistochemical detection of 3bHSD. Values are the mean ± SEM from 4 cultures. (B) RT-qPCR analysis of the effect of 106 M genistein on the mRNA levels of genes involved in steroidogenesis in Leydig cells. RNA was isolated from E12.5 testes after 3 days of culture in the presence or not of 106 M genistein. Results were calculated using the delta-delta Ct method and b-actin as reference. The mRNA levels are the mean ± SEM relative to the values of control testes that were considered equal to 1, with each sample processed in duplicate. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 versus the corresponding control value (paired Student’s t-test, n = 6).

the whole testis and specifically Leydig cells did not present apparent morphological alterations. Therefore we investigated whether the effect of genistein on steroidogenesis was due to the modulation of the expression of genes coding for proteins involved in testosterone biosynthesis. Indeed, analysis of the expression of key genes (StAR, p450scc, p450C17 and 3b-HSD) by RT-PCR showed a 40–50% reduction in the expression levels of all the genes tested, except for p450scc for which the reduction was lower, but still significant in comparison to control testes (Fig. 2B). The inhibitory effect of 106 M genistein on testosterone production by E12.5 testes was not affected by the absence of ERb, while inactivation of ERa completely suppressed the effect of genistein, demonstrating that genistein exerts its inhibiting effect via ERa (Fig. 3). Similar results were obtained when using 108 M genistein (results not shown). The effect of genistein on testosterone production was also tested in E18.5 testes, but no alteration was observed (Fig. 4). Since LH is present in vivo at this stage (O’Shaughnessy et al., 2002), we also tested the effect of genistein in the presence of oLH in the culture medium during the 3 days of culture, but the genistein effect on testosterone production was no longer observed (Fig. 4).

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Testosterone secretion (% of control)

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Fig. 3. Genistein reduces testosterone production in E12.5 fetal testes via ERa. E12.5 testes from ERb / and ERa / (/, gray bars) or wild-type mice (+/+, black bars) were cultured for 3 days in the absence (control) or in the presence of 106 M genistein. Testosterone secretion in the presence of genistein is expressed as the percentage relative to control values. Values are the mean ± SEM from 6 to 10 cultures. ⁄p < 0.05, ⁄⁄p < 0.01 versus the corresponding control value in the paired Student’s t-test. sss: p < 0.01 (unpaired Student’s t-test for different genotypes, n = 4–7).

3.2. Effect of genistein on germ cells We counted the number of gonocytes in E12.5, E18.5 and PN1 (germ cell proliferation resumes at this stage) testes after 3 days of culture in the presence of 106 M genistein. Genistein did not modify the number of gonocytes at any stage (Fig. 5).

4. Discussion We show here that short exposure to concentrations of genistein that are compatible with human consumption leads to reduction in testosterone production by E12.5 mouse testis. This is the first time that an effect of genistein on a physiological function of the fetal testis has been highlighted, because most of the previous reports focused on the effect in adult age of fetal and neonatal exposure to phytoestrogens (Sharpe et al., 2002; Tan et al., 2006; Wisniewski et al., 2003). The inhibition of steroidogenesis following incubation with genistein was limited in time as no effect was observed in cultures of E18.5 testes. This finding suggests the existence of a short period during development in which testicular steroidogenesis is sensitive to genistein. We have already reported that EDs exert different effects on fetal testosterone production depending on age, for instance estradiol or DES (Delbes et al., 2007) in the rat or for phthalates in the mouse (Lehraiki et al., 2009). This can be due to age-related modifications of the relative levels of the enzymes of the steroidogenic pathway, resulting in a

E12.5

change of the limiting enzymes and/or of the concentrations of various steroids that can interfere with the ED effects. In the rat, testicular estradiol production was incriminated in the modification of the effect of exogenous estradiol or DES (Delbes et al., 2007) and it might also be implicated in the absence of effect of genistein at late developmental stages we report here. This short time window of sensitivity can explain the lack of in vivo effects of a phytoestrogen-rich diet on steroidogenesis when measured at the end of fetal life in the mouse (Cederroth et al., 2010), and the fact that few studies have reported a decrease in testosterone production (Wisniewski et al., 2003) after exposure to phytoestrogens. However, the time window during which genistein is effective corresponds to a period when testosterone production is critical for masculinization of the reproductive system (Welsh et al., 2008). This ‘‘masculinization programming window’’ has not been defined in the mouse but, based on observations in the rat (Welsh et al., 2008), should occur within E13.5 and E17.5. Therefore, genistein may affect the masculinization program, particularly as this effect is obtained at doses commonly found in human diets (Foster et al., 2002). We then show that the genistein-induced decrease in testosterone production is not due to a reduction in the number of Leydig cells, but to the inhibition of the steroidogenic pathway. These results show that genistein has similarities with DES and estradiol in its effects on testosterone production by the fetal testis (Delbes et al., 2005). Specifically, using E13.5 mouse testes and a comparable experimental protocol, we showed that 106 M DES reduces steroidogenesis by about 20% (Delbes et al., 2005). Since, in the present study, 106 M genistein inhibited testosterone production by about 70%, genistein therefore seems to be at least as potent as DES in inhibiting testosterone production in fetal testis. This is an interesting and surprising finding because phytoestrogens are generally considered to have a weaker estrogenic activity than DES. We finally demonstrate that genistein acts through ERa, which is consistent with previous works reporting that endogenous estrogens regulate Leydig cell functions via ERa (Cederroth et al., 2007; Delbes et al., 2005). Conversely, genistein did not have any effect on gonocyte development as attested by the absence of effect on their number and morphology. This result is surprising, since both DES and estradiol have been previously reported to decrease the number of gonocytes in organ (Lassurguere et al., 2003) or cell culture (Delbes et al., 2007) of rat fetal testis at the beginning of testis development by increasing apoptosis. Moreover, ERb / neonatal mice also exhibit an increase of the number of gonocytes (Delbes et al., 2004), showing that ERb is involved in the control of gonocyte development by estrogens. Therefore, the absence of effect of genistein on germ cells is unexpected, because genistein is gen-

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Fig. 4. Genistein does not alter testosterone secretion in E18.5 testes. E12.5 and E18.5 fetal testes were cultured for 3 days in the absence (white bars) or presence of 106 M genistein (black bars). For E18.5 testes, cultures were also performed in the presence of 100 ng/ml oLH in the culture medium. Values are the mean ± SEM from 4 to 18 cultures. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001 versus the corresponding control value (paired Student’s t-test).

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Fig. 5. Genistein does not influence the number of gonocytes in fetal testes at various developmental stages. The number of gonocytes was estimated by immunostaining with an anti-MVH antibody in E12.5, E18.5 and PN1 testes after 3 days of culture in the absence (white bars) or presence (black bars) of 106 M genistein. There were no significant differences between treated and control cultures (n = 4–6).

erally considered to be a more potent agonist of ERb than ERa (Casanova et al., 1999; Kuiper et al., 1997, 1998). It has also been reported recently that genistein increases the proliferation rate of gonocytes isolated from neonatal rats (Thuillier et al., 2010). However, the authors noted that these effects involved interactions with other signaling pathways which may themselves vary in function of many factors. Activation of cell proliferation by genistein in fetal testis has also been described, but without information on the cell type involved (Montani et al., 2008). Taken together, all these results suggest that genistein by itself does not modulate germ cell number in vivo. However, it is not excluded that it might interfere mechanistically with estradiol in fetal and neonatal testis to control proliferation or apoptosis. Indeed, although fetal exposure to low doses of genistein does not alter testicular or epididymal morphology, it modifies the expression of about twenty genes (Naciff et al., 2005). Comparison of the effects of genistein on steroidogenesis and germ cells with those of other exogenous estrogens highlights that the notion of potent or weak estrogen must be considered with caution, as it has been recently pointed out (Montano et al., 2010), and could depend on the organ, but also on the age and on the effect under study. One yet unanswered major issue is whether exposure during infancy causes detrimental male reproductive effects in adulthood. Our results show that genistein has a temporary but clear deleterious effect on at least one physiological function of fetal testis very early during development and at doses corresponding to the concentrations observed in human plasma samples. Humans are exposed simultaneously to various environmental and dietary EDs, generally at low levels and exposure to genistein may affect the responsiveness and sensitivity to other EDs. Indeed, phytoestrogens are capable of altering the toxicological behavior of other EDs like methoxychlor (You et al., 2002) and synergistic (Eustache et al., 2009) or antagonistic (Lehraiki et al., 2011) effects are observed in rats treated with a low dose of genistein and the ED vinclozolin (1 mg/kg/day). In conclusion our results provide new insights into the mechanism of action of genistein in fetal testis by showing that fetal testicular testosterone production can be modulated by genistein via ERa during a short time window.

5. Conflict of interest statement All authors of this study do not have any conflicts of interest to declare.

This study was supported by the French Program on Endocrine Disruption (contract MEDD CV 05147). It was also supported by the Université Paris Diderot, by the Institut National de la Santé et de la Recherche Médicale, and by the Commissariat à l’Energie Atomique and a fellowship from the Ministère de l’Education Nationale de la Recherche et de la Technologie [237132006 to A.L.]. We thank Prof. P. Chambon for providing transgenic mice (Institut de Génétique et Biologie Moléculaire et Cellulaire, Illkirch, France) and Prof. A. Payne (Stanford University Medical Center, CA) for providing the anti-3bHSD antibody. We also thank V. Neuville, S. Leblay, and S. Rodrigues for animal care and A. Gouret for secretarial assistance.

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