ToxSci Advance Access originally published online on June 14, 2007
Toxicological Sciences 2007 99(1):234-243; doi:10.1093/toxsci/kfm160
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Developmental Changes in Testicular Sensitivity to Estrogens throughout Fetal and Neonatal Life
Laboratory of Differentiation and Radiobiology of the Gonads, Université Paris 7—Denis Diderot and CEA, DSV/iRCM/SCSR/LDRG and INSERM, Unité 566, F-92265, Fontenay aux Roses, France
1 To whom correspondence should be addressed at LDRG/SCSR/IRCM /DSV, Centre CEA, BP6, F-92265, Fontenay aux Roses, France. Fax: +33-1-46-54-99-06. E-mail: christine.levacher{at}cea.fr.
Received April 6, 2007; accepted June 11, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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There is now compelling evidence that inappropriate exposure to estrogen during fetal or neonatal life could affect adult reproductive functions because the testis is sensitive to estrogens during specific periods of its development. Therefore, we investigated the effects of exogenous estrogens on gametogenesis and steroidogenesis during fetal and neonatal testicular development in the rat. We used in vitro systems, organ cultures, and dispersed testicular cell cultures, which allow the development of fetal and neonatal germ cells (gonocytes) and Leydig cells. Exogenous estrogens inhibited testosterone production in dispersed testicular cell cultures throughout fetal life, but this inhibition was observed only in the early fetal stages in organ culture. By using an aromatase inhibitor (letrozole, Novartis Pharma AG), we showed that the inhibitory effect of exogenous estrogens on testosterone production is masked in the whole testis at later stages (20.5 days postconception) due essentially to local production of estrogens. In both systems, additions of high concentrations (10–6M) of 17ß-estradiol or diethylstilbestrol decreased the number of gonocytes during the first fetal proliferative period but not during the neonatal period. Letrozole was without effect, suggesting that the aging-related loss of responsiveness of gonocytes is not due to any aromatase activity in the gonocytes.
Key Words: testis development; fetus; neonate; gonocyte; Leydig; testosterone; estrogen.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The fetal and neonatal development of the testis from the sexually undifferentiated gonad involves a succession of events allowing the development of each testicular cell type (Jost and Magre, 1993
; Olaso and Habert, 2000). In the rat, Sertoli cells differentiate and surround the germ cells to form the seminiferous cords from 13.5 to 14.5 days postconception (dpc) and then they proliferate until 3 weeks after birth (reviewed in Pelliniemi et al., 1993). The primordial germ cells are called gonocytes after their migration from extraembryonic mesoderm to the genital ridge. The gonocytes divide actively into the seminiferous cords until 17–18 dpc and then remain quiescent until 3 dpp when they resume their mitosis (Boulogne et al., 1999b). Lastly, fetal Leydig cells differentiate in the interstitial compartment from fetal day 13.5 and produce testosterone from fetal day 15.5 (Habert and Picon, 1984; Habert et al., 2001; Warren et al., 1973). Their number increases until birth, after which they regress, and the adult Leydig cells differentiate about 2 weeks after birth.
In 1993, Sharpe and Skakkebaek (1993) had developed an "estrogen hypothesis" in which they argued that estrogen-like molecules could impair adult male fertility by acting during gonadal development and that inappropriate exposure to estrogens during fetal or neonatal life could affect adult reproductive functions. Clinical issues also support this hypothesis. Indeed, the male offspring of women treated with diesthylstilbestrol (DES), a potent synthetic estrogen, during pregnancy have been reported to show a higher incidence of reproductive disorders, such as atrophic testes and sperm abnormalities compared with placebo-exposed men (Gill et al., 1979; Glaze, 1984; Strohsnitter et al., 2001). These results are controversial since two other studies did not report any consequences in men exposed to DES in utero (Leary et al., 1984; Wilcox et al., 1995). The discrepancy between these reports could be attributed to the difference in the period of exposure during pregnancy assuming that there may be specific periods when the testis is sensitive to estrogens during fetal life. Experimental data also support the "estrogen hypothesis". In utero experiments have shown that rodents exposed to DES have abnormal testicular histology and altered adult male fertility (reviewed in Delbes et al., 2006; Sharpe, 2003). Also, in utero exposure to DES of rats at 9 and 10 dpc can induce an advance in testicular development, an abnormal differentiation of gonocytes and Sertoli cells and fetal Leydig cell hyperplasia observed from 16 dpc (Perez-Martinez et al., 1996; Yasuda et al., 1985a,b). On the other hand, by using the estrogen receptor knockout mice, we have shown that endogenous estrogens physiologically inhibit the development of the fetal and neonatal testis (Delbes et al., 2004, 2005a). The homozygous inactivation of estrogen receptor ß (ERß–/–) in mice increased the number of gonocytes by 50% in 2-day-old neonates and did not modify the numbers of Sertoli cells and Leydig cells or the levels of testicular testosterone production. The homozygous inactivation of ER
(ER–/–) had no effect on the number of gonocytes, but testosterone secretion was increased at 13.5 dpc and 2 dpp and did not involve any change in plasma gonadotropin levels.
Estrogens probably act directly on the fetal and neonatal testis, but data concerning the period of sensitivity of the testis are scattered. In organ culture of testis explanted at 13.5 and 14.5 dpc, respectively (Cupp and Skinner, 2001; Lassurguere et al., 2003), metoxychlor (a pesticide currently still used in the United States) and DES can alter the formation and architecture of the seminiferous cords. Estradiol also inhibits testosterone production in organ culture at 14.5 dpc (Lassurguere et al., 2003) and in cultured dispersed rat fetal Leydig cells at 21.5 dpc (Tsai-Morris et al., 1986). Concerning gonocytes, on one hand, we have shown that estrogens are able to inhibit the development of these cells in 14.5 dpc rat fetal testis in organ culture (Lassurguere et al., 2003) and, on the other hand, there is one report of a positive direct effect of 10–6M 17ß-estradiol (E2) on gonocyte development from 3-day-old neonates in a purified cell culture system (Li et al., 1997).
This lack of clear information prompted us to search for developmental changes in the responsiveness of the testis to estrogen action throughout fetal and neonatal life. In the present study, we analyzed the in vitro effects of estrogens using two culture models that have been described previously: an organ culture (Habert et al., 1991; Livera et al., 2006) in which the testicular architecture is conserved and the development similar to that observed in vivo and a dispersed testicular cell culture (Boulogne et al., 1999b, 2003; Gautier et al., 1997; Rouiller-Fabre et al., 1998b) in which Leydig cells and gonocytes in vitro retain some characteristics from their in vivo stage but in which paracrine factors are diluted. Using these two culture models, we have previously shown that different factors such as transforming growth factor ß (TGFß) (Gautier et al., 1997; Olaso et al., 1998), insulin-like growth factor 1 (IGF1) (Rouiller-Fabre et al., 1998b), tri-iodothyronine (Boulogne et al., 2003), and retinoic acid (Boulogne et al., 1999a, 2003; Livera et al., 2000) control the development of fetal and neonatal testis. We report here changes in gonocyte and Leydig cell sensitivity to estrogens during fetal and neonatal development.
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MATERIALS AND METHODS |
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Animals
Wistar rats (Janvier, Le Genest Saint Isle, France) were housed under a controlled photoperiod (light on 0800 h–2000 h) and were fed with commercial food (R03, Safe, Epinay-sur-Orge, France) and tap water ad libitum. Females were caged with the males for one night, and the day following overnight mating was counted as 0.5 dpc. Pregnant rats were anesthetized by an ip injection of 4 mg/100 g sodium pentobarbital (Sanofi, Libourne, France), and the fetuses were quickly removed from the uterus. Fetuses aged 14.5, 16.5, 18.5, and 20.5 days were dissected under a binocular microscope, their sex was determined by the morphology of the gonads, and the fetal testes were aseptically removed.
Natural birth occurred between day 21.5 at 1400 h and day 22.5 at 1800 h. Fetal day 22.5 was counted as 0 dpp. Three-day-old male neonates were killed by decapitation, and their testes were immediately removed. All animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health Guide).
Chemicals and Solutions
The culture medium was Ham F12/Dulbecco modified Eagle's medium (1 g/l D-glucose) (1:1 vol:vol; Gibco, Grand Island, NY) with Glutamax and contained 80 µg/ml gentamicin (Gentalline; Schering-Plough, Levallois-Perret, France). Collagenase was obtained from Serva (Heidelberg, Germany), deoxyribonuclease I (DNase I) from Sigma (St Louis, MO), and fibroblast growth factor-ß (FGF-ß) from Gibco. Ovine luteinizing hormone (oLH) (NIH.LH S19; 1.01 IU/mg) was a gift from Dr B Parlow (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). E2 and DES were from Sigma (St Louis, MO). A stock solution (1mM) was made up in ethanol and diluted in culture medium for use (5.10–6 to 10–10M). ICI 182.780 from Fisher-Bioblock (Illkirch, France) is a pure antiestrogen that suppresses estrogen activity via both ER and ERß (Sun et al., 1999, 2002). Letrozole, an aromatase inhibitor, was generously provided by Novartis Pharma AG (Basel, Switzerland).
Culture of Dispersed Testicular Cells
Testicular cells were dispersed and cultured as previously described (Boulogne et al., 1999b, 2003; Gautier et al., 1997; Rouiller-Fabre et al., 1998b). Briefly, testes were totally (16.5 dpc) or partially (20.5 dpc and 3 dpp) dissociated by enzymatic digestion (0.2 mg/ml collagenase and 0.01 mg/ml DNase) combined with mechanical disruption by repeated pipetting. For cells at 16.5 dpc, at the end of the digestion period, 30 ml of Dulbecco's PBS was added and allowed to settle for 5 min under gravity, which eliminated the remaining undigested fragments. For later stages, the settling was completed during 12 min which allowed for the collection of the interstitial cells (Leydig cells) in the supernatant and the undigested cord fragments in the pellet, which was digested again for 20 min to release the gonocytes. The different cell suspensions were collected, centrifuged for 15 min at 100 g, and the pellet was suspended in a defined volume of culture medium.
Gonocytes/Sertoli cells coculture.
Twenty fetuses at 16.5 dpc or four neonates were sacrificed. Cell suspension was mainly somatic cells (essentially Sertoli cells), and gonocytes accounted for 15–20% and 2–3% of fetal and neonatal cells, respectively (Boulogne et al., 2003). Unless otherwise stated, the cells were resuspended in culture medium containing 2 ng/ml FGF-ß and seeded (D-1) in 96-multiwell culture dishes (0.32 cm2 area; Falcon, Grenoble, France) at 70,000 cells per 100 µl per well for fetal cells and 80,000 cells per 100 µl per well for neonatal cells. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. E2, DES, or other factors (letrozole, ICI 182.780) were added after 24-h plating (D0) at the indicated concentration. On day 3 (D3), 50% of the medium was replaced by fresh medium containing fresh factors, and the culture was pursued until D5. At D5, the numbers of somatic and germ cells were obtained by trypsinization (trypsin-EDTA, Gibco) and counting the cells in a hemocytometer as previously described (Boulogne et al., 2003). Briefly, toluidine blue, a vital dye, was added (0.001%) to the cell suspension before counting to facilitate identification of gonocytes. The 5-bromo-2'-deoxyuridine (BrdU) incorporation index was measured on D3 in cells cultured in Lab-Tek chambers (Nunc, Roskilde, Denmark) as previously described (Boulogne et al., 2003).
Dispersed fetal Leydig cells.
Twenty fetuses at 16.5 dpc or seven fetuses at 20.5 dpc were sacrificed. Cell suspension contained 2–5% Leydig cells. The cells were resuspended in culture medium containing 0.5% fetal calf serum (FCS) and seeded (D1) in 96-multiwell culture dishes at 90,000 cells per 100 µl per well (Gautier et al., 1997; Rouiller-Fabre et al., 1998a). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air. After 24-h plating (D0), medium was replaced by a fresh medium without FCS and supplemented with 100 ng/ml oLH to avoid dedifferentiation of the Leydig cells. E2 or DES was added at the indicated concentration. The culture was performed for 48 h (D2). The media were changed every 24 h and kept at – 20°C for testosterone radioimmunoassay (RIA). At D2, Leydig cells were identified by cytochemical detection of 3ß-hydroxysteroid dehydrogenase activity (Steinberger et al., 1966) after freezing and counted, as previously described (Gautier et al., 1997; Rouiller-Fabre et al., 1998a).
Organ Culture
Testes were cultured on Millipore (Bedford, MA) filters (pore size 0.45 µm) as previously described (Habert et al., 1991; Livera et al., 2006). Briefly, intact testes from 14.5 dpc fetuses were placed on filters. Older testes were cut into small pieces (two and eight pieces for 16.5 and 20.5 dpc fetuses, respectively, and 16 pieces for 3-day-old neonates), and all the pieces from one testis were placed on a single filter. The filter was floated on 0.4 ml (14.5 dpc fetuses) or 1.5 ml (later stages) culture medium in tissue culture dishes and incubated at 37°C in a humidified atmosphere containing 95% air/5% CO2. The culture was performed for 72 h, and the medium was changed every 24 h. For each animal, one testis was cultured in control medium and the other testis was cultured in medium supplemented with E2 or DES or other factors as indicated. At the end of the culture period, 100 ng/ml oLH, and possibly 1mM BrdU, was added for the last 3 h of the culture. The explanted testes were fixed for 2 h in Bouin's fluid, embedded in paraffin, and cut into 5 µm sections for histological analysis. Media were kept at – 20°C for testosterone RIA.
Identification and counting of the gonocytes.
Serial sections were mounted on slides, deparaffinized, rehydrated, and stained. The gonocytes at 3 dpp were identified by immunocytochemical detection of TGFß2 as previously described (Olaso et al., 1997) (Fig. 6a). All the gonocytes in five different sections, randomly distributed through the pieces of the gonad, were counted (Crude Count [CC]). The Abercrombie formula (Abercrombie, 1946) was used to correct for any double counting resulting from the appearance of a single cell in two successive sections: TC = CC x S/(S + D) where "TC" is the true count, "S" is the section thickness (5 µm), and "D" is the true mean diameter of the gonocyte nuclei. D equals the average of the nuclear diameters measured (DM) on the section divided by 
/4 to correct for the overexpression of smaller profiles in sections through spherical particles. "DM" was measured on each testis by at least 100 random determinations using a computerized video micrometer (Histolab, Microvision Instruments, Evry, France). The TC was divided by the corresponding section area measured by a computerized video densitometer (Histolab) to determine the average gonocyte density per surface unit. This density was then multiplied by the cumulative areas of the sections from the testis to obtain the total number of gonocytes in the whole testis. All counts were done blind.
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Measurement of BrdU incorporation index.
Testes (3 dpp) were labeled with BrdU as previously described according to the manufacturer's recommendations (Olaso et al., 1998
) (Fig. 6b). The BrdU incorporation index (percentage of labeled gonocytes) was obtained by a blind counting of at least 300 gonocytes or 700 Sertoli cells on the sections.
Identification and counting of the Leydig cells.
Leydig cells were identified by immunocytochemical detection of 3ßHSD activity as previously described (Livera et al., 2000). All the Leydig cells in every 10th section of whole testis at 14.5 dpc were counted, and the Abercrombie formula was applied. For older stages, five different sections randomly distributed through the pieces of the gonad were counted and corrected using the Abercrombie formula. The number of Leydig cells per section was divided by the corresponding section area measured by a computerized video densitometer (Histolab) to determine the average density of Leydig cells per surface unit. This density was then multiplied by the cumulative areas of the sections from the testis to obtain the total number of Leydig cells in the whole testis. All counts were done blind.
Testosterone RIA
The testosterone secreted into the medium was measured in duplicate by RIA as previously described (Habert and Picon, 1984).
Statistical Analysis
All values are the means ± SEM. The significance of the difference between the mean values of the treated and untreated testes from the same animal in organ culture or from the treated and untreated dispersed cell cultures were evaluated using the Student's paired t-test.
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RESULTS |
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Effects of Estrogens on Steroidogenesis
Fetal dispersed testicular cells.
Testosterone secretion by dispersed testicular cells at 16.5 and 20.5 dpc was measured after 48 h of culture. E2 or DES treatment (10–10 to 10–6M) had a dose-dependent inhibitory effect on testosterone secretion per well (Fig. 1A) at both stages.
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Parallel to this negative effect, the number of Leydig cells, as determined after cytochemical detection of 3ßHSD activity, was significantly increased by the higher concentrations of E2, and high concentration of DES leading only to a slight and nonsignificant increase (Fig. 1B). The incorporation of BrdU into the Leydig cells was measured and was negligible even in the presence of estrogens (0–2 cells per well, data not shown).
Consequently, when expressed per Leydig cell, testosterone secretion was strongly reduced in a dose-dependent manner by E2 or DES (Fig. 1C).
Organ cultures.
Testes from fetuses at 14.5 dpc cultured for 3 days in the presence of 5.10–6M DES exhibited a significant decrease in the number of Leydig cells (Fig. 2A) and a significant decrease in whole testosterone production (Fig. 2B), the basal, or LH testosterone secretion per cell also being significantly impaired (Fig. 2C). In testes from fetuses at 16.5 dpc, no alteration of whole testosterone production was observed after DES treatment, and a slight but not significant alteration of the number of Leydig cells was noted. However, when expressed per Leydig cell, the testosterone secretion was significantly inhibited. In testes from fetal day 20.5, DES did not modify the number of Leydig cells or the testosterone production per testis or per Leydig cell.
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When the testes were cultured in the presence of 5.10–6M E2 (Fig. 3), a decrease in the number of Leydig cells was observed in testes explanted on fetal day 14.5 and not in testes from fetal day 20.5 (Fig. 3A). The testosterone production per testis was not altered at 14.5 dpc, but E2 induced a weak but significant increase at 20.5 dpc (Fig. 3B). The resulting testosterone secretion per cell presented no significant alteration at either 14.5 or 20.5 dpc (Fig. 3C).
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Effects of ICI and letrozole on testes at 20.5 dpc.
The fact that the addition of DES or E2 resulted in different effects in cell cultures and in organ culture in terms of testosterone production at fetal day 20.5 prompted us to elucidate the mechanism involved. In organ culture, at 20.5 dpc, the addition of 4µM ICI 182.780, an antiestrogen, or of 200nM letrozole, an aromatase inhibitor, significantly increased the LH-stimulated testosterone secretion on D3 (Fig. 4). Moreover, while 10–6M DES alone was unable to reduce testosterone secretion, in the presence of letrozole, 10–6M DES restored testosterone secretion to its control value. These results strongly suggest that the endogenous production of estrogens saturates the estrogen receptors in the Leydig cells at this stage, leading to insensitivity to addition of exogenous estrogens.
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Effect of Estrogens on Gametogenesis
Cocultured gonocytes/Sertoli cells.
The effect of E2 or DES was studied on cells obtained from fetal testes at day 16.5 (the earliest stage at which gonocytes survive in this culture system) and from neonatal testes at 3 dpp. These stages correspond to the two proliferative periods of the gonocytes, before and just after the quiescent phase (Boulogne et al., 1999a
, 2003).
After 5 days of culture, the number of fetal gonocytes was significantly decreased by 10–6M E2 or DES but not by 10–10 or 10–8M E2 or DES (Fig. 5). The capacity of the cells to incorporate BrdU was evaluated at D3 of culture (at the time when the cells proliferate the most actively in culture (Boulogne et al., 2003). BrdU-positive gonocytes represented 3.98 +0.86% (n = 5) of the gonocyte population in control cultures versus 4.58 + 1.09% (n = 5, ns) in E2-treated and 3.99 + 1.08 (n = 5, ns) in DES-treated cells, showing that the decrease in the number of gonocytes by estrogens was not due to a reduction in their mitotic activity. Conversely, the number of neonatal gonocytes was not modified by any treatment (10–6M E2 or DES) (Fig. 5). Lower concentrations of estrogens (10–10 or 10–8M E2 or DES) were also without effect, and 10–5M DES was toxic for the cultures (results not shown).
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The number of somatic cells was never significantly affected by E2 or DES at the fetal stage (16.5 dpc) or at the neonatal stage (3 dpp) (Fig. 5).
It has recently been shown that aromatase is present in the fetal gonocytes in pig (Haeussler et al., 2007). To test whether or not the insensitivity of the gonocytes to estrogens at 3 dpp was due to intrinsic aromatase activity, letrozole or ICI 182.780 was added to the cell cultures at 3 dpp at the same concentrations as those used for Leydig cell study. No significant modification of the number of the gonocytes or Sertoli cells was observed (Fig. 5).
Organ cultures.
The treatment of testes from 3-day-old neonates with 5.10–6M E2 or DES (Fig. 6) for 3 days did not affect the gross histology of the testis (Figs. 5a and 5b). In agreement with the results obtained in cell cultures, 5.10–6 or 5.10–8M (results not shown) E2 or DES (Fig. 6) did not modify the number of gonocytes or the incorporation of BrdU into the gonocytes at the end of the 3-day culture period. Sertoli cell mitotic activity, as attested by the percentage of BrdU-positive cells, was significantly reduced by 5.10–6M E2 but not by DES.
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DISCUSSION |
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The present data demonstrate that, during fetal and neonatal testicular development in the rat, there is a specific estrogen-sensitive period for gametogenesis, whereas steroidogenesis is constantly inhibited by estrogens.
We show here that estrogens can decrease testosterone production of fetal Leydig cells not only at 14.5 dpc, as previously reported (Lassurguere et al., 2003), but also throughout fetal life in cell culture. Inhibition of steroidogenesis in Leydig cells from rat at 21.5 dpc in a cell culture model had already been described (Tsai-Morris et al., 1986), and the present results are also in accordance with the observation that testosterone production is increased in testes of ERKO mice at 13.5 and 2 dpp (Delbes et al., 2005b). In these mice, the increase in testosterone production was due to enhanced transcription of enzymes involved in the steroidogenic pathway without any modification of plasma LH in the 2-day-old neonates. However, while testosterone secretion was constantly inhibited by addition of E2 and DES in cell culture at a concentration as low as 10–10M, the whole testis, in organ culture, exhibited temporal changes in estrogen sensitivity. In organ culture, the inhibition of Leydig cell differentiation by 5.10–6M E2 or DES observed in the fetal testis at 14.5 dpc disappeared afterward. This observation suggested the existence of an intratesticular mechanism able to mask the exogenous estrogen effect in the whole testis that is not present in the dispersed cell culture model. One candidate could be the presence in the fetal testis of low levels of binding proteins (Becchis et al., 1996), which have been shown to exert a negative control on estradiol action in MCF-7 cells (Fortunati et al., 1996). However, we have reported discrepancies between the two culture models concerning the effects on steroidogenesis of exogenous factors such as TGFß1 (Gautier et al., 1997) and IGF1 (Rouiller-Fabre et al., 1998b). In all cases, it appeared that in organ culture the cells are less sensitive to exogenous agents, probably because of a still high intratesticular concentration of these factors. Fetal testis produces endogenous estradiol from 15 to 17.5 dpc in the mouse, as suggested by the expression of aromatase mRNA (Greco and Payne, 1994). At these stages in the rat, it is likely that estradiol is produced by the Sertoli cells since the aromatase activity is stimulated by FSH (Abney, 1999; Rouiller-Fabre et al., 1998a; Weniger and Zeis, 1988). In the rat, the E2 concentration is about 0.5nM in the testis and in the serum at 18/20 dpc, but this increases after birth (Habert and Picon, 1984). We show here that the inhibition of endogenous testicular estrogen production by letrozole, an aromatase inhibitor, leads to an increase in testosterone production by the testis, probably by removing the inhibition from endogenous estrogens, and that, in the presence of letrozole, DES is able to inhibit testosterone production, as observed in cell culture. The involvement of endogenous estrogens was confirmed by the fact that ICI 182.780, an antagonist of ER, also induces an increase in testosterone production at 20.5 dpc, whereas it was without effect at 14.5 dpc (Lassurguere et al., 2003). Thus, in the fetal testis, estrogens are able to inhibit testosterone production by inhibiting the steroidogenic pathway (Delbes et al., 2005b; Tsai-Morris et al., 1986), but if the level of endogenous estrogens produced inside the testis becomes too high, exogenous estrogens are therefore without effect. Nevertheless, this does not exclude the existence of other intratesticular mechanisms responsible for the insensitivity of the whole rat testis to exogenous estrogens during late fetal life and does not exclude other sensitive end points that were not measured in this study. Moreover, there may be species specificity since, in the mouse, testosterone production can be inhibited by DES in organ culture (Delbes et al., 2005b).
The regulation of Sertoli cell proliferation by estrogens is not clear and the mechanisms involved remain to be elucidated. Indeed, in organ cultures, DES was able to reduce the number of Sertoli cells at 14.5 dpc (Lassurguere et al., 2003) by a mechanism that is probably not mediated by ER, and at 3 dpp E2, but not DES, slightly reduced their proliferation (present data). In contrast, estrogens did not modify the number of Sertoli cells in cell culture, probably due to the difference in experimental models. Therefore, during early fetal life, a modification of the number of gonocytes can occur without any modification of Sertoli cell number which is in favor of a direct action of estrogens on gonocytes during this period, as previously suggested (Lassurguere et al., 2003) and not through an alteration of the Sertoli cells, although some alterations of other functions of these cells cannot be excluded.
It must be pointed out that there exist some differences in the effect of DES and estradiol on the proliferation of Sertoli cells in organ culture and of Leydig cells in cell culture. These molecules have been already shown to have both common and individual testicular gene expression pattern in the mouse testis (Adachi et al., 2004) and to produce distinctly different activation of extracellular regulated kinases and of Phosphoinisitide-3 kinase (PI3K) via membrane-initiated signaling pathways in pituitary tumor cell line (Bulayeva et al., 2004).
We show here that estrogens are deleterious for gonocytes during the fetal proliferative period (16.5 dpc) since E2 and DES reduced their number, probably due to increased apoptosis. This is in accordance with our previous results showing a negative effect of estrogens on gonocyte development at 14.5 dpc i.e., at a stage of the same fetal period of proliferation/apoptosis (Lassurguere et al., 2003). In two culture systems, we also show here for the first time that gonocytes are insensitive to estrogens on postnatal day 3 i.e., during neonatal proliferation. Thus, we clearly define an estrogen-sensitive period and a nonestrogen-sensitive period of gonocytes concerning the control of their number. A previous study reported a positive effect of E2 on gonocytes at 3 dpp in vitro (Li et al., 1997) at the only concentration of 1µM. The discrepancy with our present data could be explained by the difference in experimental model since they cultured isolated gonocytes in the presence of fetal bovine serum. In our study, the culture medium did not contain serum, and the gonocytes were cultured with somatic cells and could interact with them. The reason for the insensitivity of gonocytes to estrogens during the neonatal wave of proliferation is not known and remains to be investigated. A recent study in the pig pointed out that aromatase is expressed in the gonocytes during fetal life (Haeussler et al., 2007), suggesting that a mechanism similar to the one we demonstrated for the Leydig cell insensitivity at the level of the whole testis could take place inside the gonocytes themselves. But the absence of effect of letrozole or ICI 182.780 on the number of gonocytes strongly suggests that the reason for this insensitivity should be sought elsewhere. We can hypothesize that fetal and neonatal gonocytes have different properties as, for example, they go from a hypomethylated to a hypermethylated state (Anway and Skinner, 2006; Coffigny et al., 1999). It is important to note that, even during early development, when estrogens have a deleterious effect on gonocyte number, this effect is obtained only with concentrations of estrogens higher than those required to modify the steroidogenic activity of the Leydig cells in cell cultures (present data) and in organ cultures (Lassurguere et al., 2003). This suggests that the mechanism of estrogen action could be different in the germ cells and Leydig cells. This is in accordance with the fact that endogenous estrogens regulate the number of gonocytes via ERß and testosterone secretion via ER (Delbes et al., 2005a). Alterations in StAR and P450c17 testicular gene expression without alterations of markers of testicular cell lineage differentiation have also been reported in 18.5 dpc mice after in utero exposure to DES (Guyot et al., 2004). However, the fact that estrogens do not affect the number of gonocytes at 3 dpp does not mean that they cannot alter other parameters of their development/differentiation.
Among the numerous studies concerning the boys born to women treated with DES during their pregnancy between 1950 and 1970, some reported alterations in the quality of the sperm (Glaze, 1984; Strohsnitter et al., 2001), whereas others reported no change (Wilcox et al., 1995). A recent analysis of these epidemiological studies (Storgaard et al., 2006) specifies that the negative effect of DES on sperm count was observed only if DES was given at high dose during the first semester of pregnancy. Our present observation of the existence of an estrogen-sensitive period of gonocyte development in vitro in the rat could explain the effects observed in vivo in humans.
These results show that there are windows of testicular sensitivity to estrogens during fetal and neonatal development, depending on cell type and perhaps on species. This study also suggests that intratesticular architecture and dialogue are able, in some cases, to "protect" the testis against exogenous endocrine disruptors, at least for those that could act via ER.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Université Denis Diderot-Paris 7, Institut National de la Santé et de la Recherche Médicale, Commissariat à l'Energie Atomique, and Institut National de la Santé et de la Recherche Médicale Project Environnement et Santé. Ministère de l'Education Nationale de la Recherche et de la Technologie and the Association pour la Recherche sur le Cancer to G.D.
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ACKNOWLEDGMENTS |
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We thank V. Neuville and P. Flament for animal care, A. Gouret for secretarial assistance, Dr A. Payne for generously providing the anti-3ßHSD antibody, and Novartis Pharma AG (Basel, Switzerland) for generously providing letrozole.
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REFERENCES |
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