* GERM-INSERM U.435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France, and
INSERM-CEA U.566, Université Paris 7, Bat 5 du DRR, BP6, 92265 Fontenay-aux-Roses Cedex, France.
Received December 18, 2002; accepted February 14, 2003
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ABSTRACT |
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Key Words: diethylstilbestrol; estradiol; anti-estrogen; fetal testis; Sertoli cells; Leydig cells; gonocytes; organ culture.
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INTRODUCTION |
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These observations clearly show that molecules with estrogenic properties can have deleterious effects on fetal testicular development. This and other findings led to the concept that environmental substances that mimic, antagonize, or alter the metabolism of hormones, named endocrine disruptors, exert their deleterious effects on the male reproductive system by interfering with key physiological events during the prenatal period (McLachlan, 2001; ODonnell et al., 2001
; Sharpe, 1993
; Sharpe and Skakkebaek, 1993
). However, although a large amount of international research has taken place on the endocrine disruption issue over the last decade, the precise cellular and molecular mechanisms underlying the action of estrogen on the fetal testis are still largely obscure. In particular, it is still unknown whether the effects of estrogens or xeno-estrogens result (1) from the disruption of the general physiology/endocrinology of the pregnant mother; (2) from altered fetal gonadotropin secretion, as Sertoli cells and Leydig cells are targets for the pituitary gonadotropin luteinizing hormone (LH) and follicle-stimulating hormone (FSH), respectively (Atanassova et al., 1999
, 2000
; Sharpe et al., 1998
); (3) from direct estrogen receptor-mediated effects on fetal Leydig cells, Sertoli cells, and germ cells, as these cells express either estrogen receptors ß (ER-ß) (Jefferson et al., 2000
; Saunders et al., 1998
; van Pelt et al., 1999
) or
(ER-
) (Fisher et al., 1997
), or both (ODonnell et al., 2001
); or (4) from both gonadotropin hormone-mediated effects and direct actions (ODonnell et al., 2001
).
In addition to altering the testes in utero, DES and estrogens cause a high rate of cryptorchidism in males born to women and mice treated during pregnancy (Abney, 1999; Gill et al., 1979
; Nef et al., 2000
; Newbold, 1995
; Toppari et al., 1996
). Furthermore, a significant rate of abnormalities in the anatomical structures involved in testicular descent and positioning has been observed in ER-
knockout mice (Couse and Korach, 1999
; Donaldson et al., 1996
), which further emphasizes the role of estrogens in the mechanisms that control testicular descent during development. Testicular malpositioning is classically associated with disrupted spermatogenesis; therefore, it is not possible to distinguish between any testicular changes that result from the altered migration of the fetal testis and any changes resulting from ER-mediated effects and/or via gonadotropin-mediated effects. Furthermore, we do not know whether the estrogen-induced changes in sperm output in adulthood result from the disruption structure and function of the efferent ducts, as is the case in ERKO mice (Hess et al., 1997
).
These questions prompted us to use an organotypic culture model coupled to morphological and functional methods to analyze the development of the fetal testis previously developed by one of our laboratories to investigate the mechanisms of action of gonadotropin hormones and regulatory factors on the fetal rat testis (Habert et al., 1991; Lecerf et al., 1993
; Livera et al., 2000
, 2001
; Olaso et al., 1998
). This organ culture system preserves the testicular architecture and the intercellular communications, and allows the different testicular cell types to develop as in vivo. Experimentally, it allows a time- and a dose-controlled exposure to the assayed factor directly to the testis. In the present study, we applied the same experimental approaches to study the effects of DES and estradiol on the in vitro development of fetal testicular somatic and germ cells.
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MATERIALS AND METHODS |
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Chemicals and solutions.
The culture medium used was phenol red-free M199 (Life Technologies, Cergy Pontoise, France) supplemented with gentamycin (50 µg/ml; Life Technologies, Cergy Pontoise, France) and fungizone (2.5 µg/ml; Life Technologies, Cergy Pontoise, France). Ovine (o)LH (NIH.LH S19; 1.01 NIH.LH.S1 U/mg) was a gift from Dr. A.F. Parlow (NIDDK, Bethesda, MD). Recombinant human (h) FSH was (12,000 IU/mg) was a gift from B. Mannaerts (Organon International, Oss, the Netherlands).
Seventeen ß-estradiol (E2) and diethylstilbestrol (DES) were purchased from Sigma (St Louis, MO), and ICI 182.780 was purchased from Fisher Bioblock Scientific (Illkirch, France). ICI 182.780 is pure anti-estrogen that suppresses estrogen activity both via ER- and ER-ß (Sun et al., 1999
, 2002
). The anti-3ß-hydroxysteroid dehydrogenase (anti-3ßHSD) antibody was generously provided by Drs. G. Defaye and E. Chambaz (Grenoble, INSERM, France), and the GATA-4 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Culture Procedure.
Testes were cultured on Millipore filters (pore size: 0.45 µm) (Bedford, MA), as previously described (Habert et al., 1991). Briefly, each intact 14.5-d.p.c. fetal testis was removed with the adjacent mesonephros and placed on a filter. The filter was floated on 0.4 ml of medium in tissue culture dishes and incubated at 37°C in a humidified atmosphere containing 95% air/5% CO2 for 1, 2, or 3 days (D1, D2, D3). The medium was changed every 24 h. The response to E2 or DES was measured by comparing one testis cultured in medium containing E2 or DES with the other testis from the same fetus cultured in medium alone (control).
In some experiments, 100 ng/ml of oLH and 1 mM of 5-bromo-2'-deoxyuridine (BrdU; Amersham, Buckinghamshire, England) or 200 mUI/ml of recombinant FSH were added to the dishes for the last 3 h of the culture. For cellular analyses, the whole explant was fixed for 2 h in Bouins fluid, except for samples for TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end-labeling) assessment, which were fixed with buffered formaldehyde at 4%. All the explants were embedded in paraffin, and 5 µm sections were cut.
When assessing cyclic adenosine 5'-monophosphates (cAMP), three testes instead of one were cultured together on the same filter for 72 h, as described later, to obtain detectable values.
Quantification of gonocytes.
Gonocytes were counted as previously described (Livera et al., 2000; Olaso et al., 1998
). Briefly, all the serial sections from one explant were cut, and 1/3 of them were mounted on slides, deparaffinized, rehydrated, and stained with Massons hemalun and eosin. The gonocytes were identified by their large, spherical, lightly stained nuclei containing fine chromatin granules and two or more globular nucleoli, and by their clearly visible cytoplasmic membrane. All of the gonocytes in each section were counted and added together to give the total count (TC), which was then multiplied by 3 to give the crude count (CC) of gonocytes per testis. The Abercrombie formula 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 and S is the section thickness (5 µm) (Abercrombie, 1946
). D is the average nuclear diameter measured on the section (DM) divided by
/4 to correct for the overrepresentation of smaller profiles in sections through spherical particles. In each testis studied, DM was measured from at least 100 random determinations, using a micrometer eyepiece and calibrated with an object micrometer on the microscope plate. All counts and measurements were done at random and cross-checked periodically by a second experimenter.
Quantification of Sertoli cells.
Sertoli cells were identified by immunostaining for a transcription factor named GATA-4 that stains fetal Sertoli cells and Leydig cells (Viger et al., 1998). Deparaffinized sections were rehydrated and incubated for 1 h in the presence of the anti-GATA-4 antibody (200 µg/ml) in a humidified chamber at room temperature, and bound primary antibodies were revealed with a biotinylated rabbit anti-goat secondary antibody (Rockland, Gilbertsville, PA) and an avidin-biotin-peroxidase complex (DAKO, Glostrup, Denmark). Slides were stained with DAB (3,3'-diaminobenzidine, Dako, Carpinteria, CA). Sections were rinsed in PBS between each step. All the GATA-4-positive cells in every third section were counted, and the Abercrombie formula was applied.
Quantification of Leydig cells.
Leydig cells were identified by immunohistochemical detection of 3ßHSD activity, as follows (Livera et al., 2000): deparaffinized sections were rehydrated, then incubated overnight with anti-3ßHSD antibody (1:400) in a humidified chamber at 4°C. Bound primary antibodies were revealed with a biotinylated goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) and the avidin-biotin-peroxidase complex (DAKO, Glostrup, Denmark). Peroxidase was visualized with 3,3'-diaminobenzidine (Dako, Carpinteria, CA). The sections were rinsed in PBS between each step. All the Leydig cells in every third section of the whole testis were counted, and the Abercrombie formula was applied.
Measurement of the BrdU incorporation index.
Cultures of 14.5-d.p.c. testes were labeled with BrdU (labeling reagent diluted 1:100, according to the instructions for the cell proliferation kit; Amersham, Buckinghamshire, England) during the last 3 h of culture, as previously described (Livera et al., 2000; Olaso et al., 1998
). We used immunohistochemistry to detect the incorporation of BrdU into proliferating cells according to the manufacturers recommendations. Briefly, randomly chosen sections were mounted and incubated individually with 0.3% H2O2 in methanol at 20°C for 30 min to inactivate endogenous peroxidases, then in a mouse anti-BrdU monoclonal antibody (cell proliferation kit, Amersham) at 20°C for 1 h. The antibody bound to the nuclei was detected by a peroxidase-linked anti-mouse IgG (cell proliferation kit, Amersham). Finally, slides were stained with DAB (cell proliferation kit, Amersham). The BrdU incorporation index (percentage of cells showing a clear positive immunoreaction to BrdU) was obtained by counting at least 1000 gonocytes or Sertoli cell nuclei, with the experimenter blinded to the treatment group.
Measurement of the DNA fragmentation index.
Apoptotic cells were detected in situ by use of the TUNEL method previously described (Livera et al., 2000; Olaso et al., 1998
). Randomly chosen sections were incubated successively in hydrogen peroxide, permeabilizing solution (0.1% Triton X-100 in 0.1% sodium citrate) and buffer containing TdT at 0.6 U/µl (Boehringer Mannheim, Indianapolis, IN) and fluorescein-dUTP (Boehringer Mannheim). The sections were incubated with an anti-fluorescein antibody conjugated to peroxidase (Boehringer Mannheim), stained with DAB, and counterstained by a brief immersion in Massons hemalun. Positive controls were incubated with deoxyribonuclease I (100 µg/ml) (Boehringer Mannheim) for 10 min at 20°C to induce DNA strand breaks. Negative controls were incubated without TdT. Sections were rinsed in PBS between each step. The DNA fragmentation index (the percentage of cells with a clear positive TUNEL staining) was obtained by counting at least 1000 gonocytes or Sertoli cell nuclei, with the experimenter blinded to the treatment group.
Measurement of testosterone production.
The testosterone secreted into the medium was measured in duplicate by radioimmunoassay (RIA), as previously described (Habert et al., 1991). No extraction or chromatography was performed because 17ß-hydroxy-5
-androstane-3-one, the only steroid that cross-reacts significantly with testosterone (64%), is secreted in minute amounts by the fetal rat testis (Habert and Picon, 1984
).
Measurement of cAMP.
Cyclic AMP production was estimated after 72 h of culture by incubating three testes for 3 h with fresh medium containing 1 mM isobutylmethylxanthine (IBMX) (Sigma, St. Louis, MO) in the presence or absence of 200 mUI/ml recombinant hFSH (Livera et al., 2001). The media were collected and acetylated, and RIAs were performed for cAMP, as previously described (Begeot et al., 1988
).
Statistics.
All values are the means ± SEM. The significance of the differences between the mean values of the treated and untreated controls was evaluated using a Wilcoxons test.
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RESULTS |
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In the presence of DES, the mitotic and apoptotic indexes of gonocytes in 14.5-d.p.c. testes were evaluated after 2 days of culture (Figs. 3B and 3C
). The percentage of BrdU-labeled gonocytes had dropped by 22% with 4 x 10-6 M DES (Fig. 3B
). Furthermore, the number of TUNEL-positive gonocytes in DES-treated testes was 226% of the control values (Fig. 3C
).
Effect of DES on the Number of Sertoli Cells
The number of Sertoli cells was counted at D1 or D3 in cultures supplemented with DES. At 4 x 10-6 M, DES had no effect on the number of Sertoli cells on D1. In contrast, a 30% decline was observed at D3 (Fig. 4A). At 4 x 10-8 M, DES reduced the total number of Sertoli cells by 17% on D3. This decrease was not prevented by co-incubation in the presence of 4 x 10-8 M DES and 4 x 10-6 M ICI 182.780 (Fig. 4A
).
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Effect of DES on Sertoli Cell Activity
FSH-stimulated cAMP production was used as a specific measurement of Sertoli cell activity because Sertoli cells are the only testicular cell type that express the FSH-receptor. After 3 days in culture, FSH-stimulated cAMP production was inhibited by 36% in the presence of 4 x 10-6 M DES. In contrast, basal cAMP concentrations were not significantly altered (Fig. 5).
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DISCUSSION |
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We used an organ culture system and previously validated methods (Habert et al., 1991; Lecerf et al., 1993
; Livera et al., 2000
, 2001
; Olaso et al., 1998
) to show that estrogens have a time- and dose-dependent negative effect on the number and activity of the three main cell types in the testis. The chosen concentrations of E2 ranged from 4 x 10-10 M, which is close to the Kd values of ER-
and -ß (Kuiper et al., 1997
), to 4 x 10-6 M, which alters the proliferation of purified gonocytes in culture (Li et al., 1997
). The concentrations of DES were adjusted according to E2 concentrations for comparison. The particularly great work load associated with the techniques used in our study led us to select a number of experiments in which full dose- and time-dependent effects of E2 and DES were studied and in which the antagonizing action of ICI 182.780, a pure anti-estrogen (Parker, 1993
), was used to demonstrate possible ER-mediated effects.
A previous study involving an organotypic system and explanted 13.5-d.p.c. rat fetal testes suggested that 1 x 10-6 M estradiol inhibits seminiferous cord formation (Cupp and Skinner, 2001). We found that 4 x 10-6 M estradiol significantly decreased the total number of gonocytes in seminiferous cords, which could easily be characterized. This discrepancy between the observations of Cupp and Skinner (2001)
and our observations may result from the difference in the age of the explanted fetal gonad (14.5 d.p.c. vs. 13.5 d.p.c.) or from different morphological approaches (global observation of the gonad by transillumination vs. a histological study). Alternatively, the effects of estrogens may change according to the developmental stage of the testis, as is the case for GnRH (Habert et al., 1991
), TGFß (Olaso et al., 1998
), and retinoids (Livera et al., 2000
). The effects of DES observed in our study were dose- and time-dependent. Gonocytes were the first testicular cell type to be affected and the most affected cell type: the number of gonocytes decreased by 80% at the highest concentration of DES after 3 days of culture in a background of alteration of the fetal gonad integrity. After just 1 day in culture, the number of gonocytes decreased by 31 % in the DES-treated groups, whereas the number Sertoli cells was not affected. Of note is that, when rats were treated by others with DES in the neonatal period, the total volume of germ cells in the adult was also found to decrease, and the rate of germ cell apoptosis was found to increase (Atanassova et al., 1999
). We demonstrated that, whereas the E2-induced drop in the gonocyte number uniquely results from decreased gonocyte proliferation, the DES-induced drop in this number was due to both increased apoptosis and decreased gonocyte proliferation. Gonocyte response to estrogens was rapid (as soon as D1) and sensitive (effective concentration at 4 x 10-8 M DES). Furthermore, the fact that the addition of ICI 182.780 prevented the decrease in gonocyte number shows that the action of DES on the fetal germ cells involves estrogen receptor(s). Indeed, ER-ß (Jefferson et al., 2000
; van Pelt et al., 1999
) and ER-
(Rauch and Jégou, unpublished data) are both present in rat gonocytes.
Like DES, we found that E2 also had a deleterious effect on the population of gonocytes. However, the amplitude of this effect was consistently lower than that of DES, which is compatible with the established potencies of these two molecules (Kuiper et al., 1997). This finding is contradictory with the findings obtained by Li et al. (1997)
, who found that E2 had either no effect at 10-7 M and 10-5 M or a mitogenic effect at 10-6 M on a population of gonocytes isolated from 3-day-old rats. However, the culture conditions used and the age of animals when the gonocytes were studied by Li et al. were radically different to those developed here, in which the fetal gonocytes were maintained in their anatomical environment, because Li et al. incubated pure gonocytes from 3-day-old pups (Li et al., 1997
).
In addition to its effects on gonocytes, DES also decreased the Sertoli cell population on D3 of culture. However, the proportion of cells lost was lower than that of gonocytes. Sharpe and collaborators also found that the number of rat Sertoli cells decreased after the in vivo neonatal administration of DES (Sharpe et al., 1998). Although these authors used a GnRH antagonist to discriminate between the effects of FSH and LH, and those of DES, they could not unequivocally disentangle the possible direct effects of estrogens on testicular cells from effects due to altered gonadotropin levels, testicular descent, and/or the possible dysfunction of the testicular excurrent duct system. Here we demonstrate that, in contrast to gonocytes, the decrease in Sertoli cell number results from a decrease in the proliferation of these cells, rather than from the induction of apoptosis. Another major difference with gonocytes is that ICI 182.780 did not prevent the effects of DES on Sertoli cells. Thus, it may be that the action of estrogens on Sertoli cell number is not directly mediated by ER. It is noteworthy that fetal Sertoli cells express only ER-ß (Saunders et al., 1998
), whereas Leydig cells express both ER-
(Fisher et al., 1997
) and ER-ß (ODonnell et al., 2001
), as do gonocytes (ER-
: our unpublished results; ER-ß: ODonnell et al., 2001
). Therefore, the origin of the estrogen-induced Sertoli cell depletion remains to be investigated further.
Our experiments also show that the 14.5-d.p.c. control testes cultured for 3 days responded well to FSH, in terms of cAMP production. This confirms our previous results (Livera et al., 2000; Livera et al., 2001
) and is consistent with the finding that FSH receptors are expressed and functional in the testis from 15.5 d.p.c. onward (Lecerf et al., 1993
; Rannikki et al., 1995
). DES had no effect on basal cAMP concentrations but decreased the acute FSH-induced cAMP production by 36%. However, when adjusted in function of the reduction in the Sertoli cell number (30%), this decrease was not significant. This result is important because it shows that DES does not affect the FSH-regulated activity, at least as far as the cAMP pathway is concerned.
One of the major observations made in the present study was that the number of gonocytes decreased before the number of Sertoli cells decreased. Given that the function of the Sertoli cells appeared to be unaffected, this is not compatible with the hypothesis that the estrogen-induced changes in the fetal Sertoli cell proliferation are an essential determinant in the estrogen-induced decrease in the germ cell complement (Sharpe and Skakkebaek, 1993). However, the fact that the Sertoli cell population decreased after the gonocyte population may have greatly amplified the direct negative effects of estrogens on germ cells. It is noteworthy that the decrease in Sertoli cell number could not be accounted for by the decrease in the gonocyte number, because when the latter was abolished by ICI 182.780, the Sertoli cell number remained low in the DES-treated gonads.
The fetal Leydig cells were the third cell type found to be affected by E2 and DES. These estrogens reduced both basal and LH-stimulated testosterone production. Enzymatic experiments should be developed to understand the mechanisms underlying these inhibitory effects. However, it is noteworthy that our results are in line with those of earlier studies showing that estradiol decreases testosterone production by cultured rat fetal Leydig cells by reducing 17 alpha-hydroxylase/17,20 desmolase activity (Tsai-Morris et al., 1986). Our data are also consistent with the results of a study by Majdic et al. (1996)
, showing that the in vivo treatment of rat fetuses with DES or 4-octyphenol, a putative environmental estrogen, on days 11.5 and 15.5 of pregnancy reduced the expression of testicular P450C17alpha on day 17.5. One of the novel findings of our study is that just 4 x 10-10 M DES can affect LH-induced testosterone production. Furthermore, the addition of ICI 182.780 prevented the inhibitory effect of DES on basal and LH-induced testosterone production, confirming that the action of DES on the fetal Leydig cells involves estrogen receptors, which have been found in these cells (Fisher et al., 1997
; Saunders et al., 1998
; van Pelt et al., 1999
). Our study also revealed that DES reduces the number of fetal Leydig cells. Our culture system allows the differentiation of a large number of Leydig cells as the number of Leydig cells per testis increases from 50 at the time of explantation to 3000 after 3 days in culture (Livera et al., 2000
). Leydig cells differentiate from mesenchymal cells and do not enter mitosis (Boulogne et al., 1999
). Therefore, our results suggest that DES impairs the process by which Leydig cells differentiate from mesenchymal cells. However, they cannot exclude the possibility that DES also increases the apoptosis of Leydig cells, even though we never observed apoptosis in this cell type (Boulogne et al., 1999
). An in vivo study by Majdic et al. (1996)
suggested that DES does not affect the number of fetal Leydig cells. This discrepancy may be due to the fact that Majdic et al. counted the "apparent" number of these cells, whereas we counted the total number of Leydig cells. Given that the functions of seminiferous tubules and Leydig cells are interrelated in the adult testis (Jégou et al., 1999
; Saez, 1994
), we cannot exclude the possibility that the marked changes observed in the seminiferous cords affect Leydig cell number and activity. Conversely, the drop in Leydig cell number and activity probably amplifies the E2/DES-induced alteration of the seminiferous tubule compartment.
In conclusion, our results reveal that E2 and DES can induce profound alterations in the architecture and activity of the fetal testis. In this context, the organotypic culture of the fetal testis used here provides a potential technical contribution, given the current development of tools and the fact that end-points of steroid and xeno-hormone action are not available in men for risk assessment approaches.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at GERM-INSERM U.435, Campus de Beaulieu, Université de Rennes I, 35042 Rennes Cedex, Bretagne, France. Fax: +33-223-23-5055. E-mail: bernard.jegou{at}rennes.inserm.fr.
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