Time- and Dose-Related Effects of Estradiol and Diethylstilbestrol on the Morphology and Function of the Fetal Rat Testis in Culture

Julie Lassurguère*, Gabriel Livera{dagger}, René Habert{dagger} and Bernard Jégou*,1

* GERM-INSERM U.435, Université de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, Bretagne, France, and {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms underlying the action of estrogens in the fetal testis are still largely obscure. In particular, whether this action is direct or indirect remains largely unexplored. This study was aimed at investigating the effect of estradiol (E2) and diethylstilbestrol (DES) on the testis from 14.5-day-old rat fetuses in culture, at concentrations ranging from 4 x 10-10 M (Kd of E2 for the estrogen receptors [ER]: 1–4 x 10-10 M) to 4 x 10-6 M (concentration previously shown in the literature to affect in vitro gonocyte proliferation). Exposure to DES and E2 decreased gonocyte number, the effects of DES being much more drastic than those of E2. Gonocyte number decreased in a concentration-dependent manner (day 3: -5%, -16%, and -80% at 4 x 10-10 M, 4 x 10-8 M, and 4 x 10-6M of DES, respectively), as well as in a time-dependent manner (at 4 x 10-6 M DES: -31% on day 1, -60% on day 2, and -80% on day 3). This was due to a decrease in the gonocyte mitotic index and a dramatic increase in apoptosis. Importantly, in the presence of the anti-estrogen ICI 182.780 (ICI), the effect of DES was abolished. Sertoli cell number subsequently decreased (day 3), although the rate of apoptosis did not increase. These changes were less dramatic than those observed with gonocytes and were due to a decrease in Sertoli cell proliferation, which was not antagonized by ICI. Addition of 4 x 10-6 M DES had no effect on basal Sertoli cell cyclic adenosine 5'-monophosphate (cAMP) levels or on follicle-stimulating hormone (FSH)-stimulated cAMP production after adjustment for Sertoli cell number. Finally, estrogens reduced both Leydig cell number (–26% on day 3, 4 x 10-6 M DES) and basal and luteinizing hormone (LH)-stimulated testosterone production. The latter effects were antagonized by ICI. In conclusion: 1) E2 and DES induce alterations in the germ line and in somatic cells; 2) gonocyte alteration was the first event detected, and the action of estrogens at this level was mediated by ER, as is the case in Leydig cells; and 3) these data should help us to understand estrogen effects on the fetus and should be considered in the context of the debate on environmental estrogens.

Key Words: diethylstilbestrol; estradiol; anti-estrogen; fetal testis; Sertoli cells; Leydig cells; gonocytes; organ culture.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The exposure of pregnant women to the nonsteroidal estrogen diethylstilbestrol (DES) can result in major testicular abnormalities, including Leydig cell hyperplasia, capsular induration, and hypotrophic testes, and can also result in a deterioration in sperm quantity and quality (Gill et al. 1977Go, 1979Go; Stillman, 1982Go; Toppari et al., 1996Go). In utero experiments have revealed that DES-exposed mice present various testicular abnormalities and poor semen quality (Fielden et al., 2002Go; McLachlan et al., 1975Go; Newbold, 1995Go; Newbold and McLachlan, 1985Go). The administration of estradiol or ethinyl estradiol to pregnant mice also alters Leydig cell morphology and function (Abney, 1999Go; Hadziselimovic and Girard, 1977Go), increasing the rate of cryptorchidism, disrupting Sertoli cell and gonocyte numbers (Yasuda et al., 1985Go), and possibly increasing the risk of testicular teratoma (Walker et al., 1990Go).

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, 2001Go; O’Donnell et al., 2001Go; Sharpe, 1993Go; Sharpe and Skakkebaek, 1993Go). 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., 1999Go, 2000Go; Sharpe et al., 1998Go); (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., 2000Go; Saunders et al., 1998Go; van Pelt et al., 1999Go) or {alpha} (ER-{alpha}) (Fisher et al., 1997Go), or both (O’Donnell et al., 2001Go); or (4) from both gonadotropin hormone-mediated effects and direct actions (O’Donnell et al., 2001Go).

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, 1999Go; Gill et al., 1979Go; Nef et al., 2000Go; Newbold, 1995Go; Toppari et al., 1996Go). Furthermore, a significant rate of abnormalities in the anatomical structures involved in testicular descent and positioning has been observed in ER-{alpha} knockout mice (Couse and Korach, 1999Go; Donaldson et al., 1996Go), 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., 1997Go).

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., 1991Go; Lecerf et al., 1993Go; Livera et al., 2000Go, 2001Go; Olaso et al., 1998Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and sample collection.
Timed pregnant female Sprague-Dawley rats were purchased from Elevage Janvier (Le Genest Saint Isle, Laval, France). The rats were anesthetized by an ip injection of 40 mg/kg sodium pentobarbital (Sanofi-Synthélabo, Libourne, France) 14.5 days postcoïtum (d.p.c.). At that age, the seminiferous tubules have became clearly recognizable, and gonocytes have entered their exponential phase of proliferation. The testes were removed from the fetuses under a binocular microscope, then immediately explanted in vitro.

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-{alpha} and ER-ß (Sun et al., 1999Go, 2002Go). 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., 1991Go). 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 Bouin’s 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., 2000Go; Olaso et al., 1998Go). Briefly, all the serial sections from one explant were cut, and 1/3 of them were mounted on slides, deparaffinized, rehydrated, and stained with Masson’s 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, 1946Go). D is the average nuclear diameter measured on the section (DM) divided by {pi}/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., 1998Go). 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., 2000Go): 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., 2000Go; Olaso et al., 1998Go). We used immunohistochemistry to detect the incorporation of BrdU into proliferating cells according to the manufacturer’s 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., 2000Go; Olaso et al., 1998Go). 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 Masson’s 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., 1991Go). No extraction or chromatography was performed because 17ß-hydroxy-5{alpha}-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, 1984Go).

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., 2001Go). The media were collected and acetylated, and RIAs were performed for cAMP, as previously described (Begeot et al., 1988Go).

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 Wilcoxon’s test.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histological Effects of E2 and DES on the Fetal Testis
A histological analysis was performed after culturing testes from 14.5-d.p.c. rat fetuses for 1, 2, or 3 days (D1, D2, or D3) with DES, E2, or alone (control). When rat fetal testes were cultured in control medium, the integrity of seminiferous cord architecture was maintained (Figs. 1AGo and 1CGo). DES (4 x 10-8 M and 4 x 10-10 M) and E2 (4 x 10-6 M) had no obvious effect on testis morphology by D3 (data not shown). Some gonocytes had disappeared by D1 following treatment with 4 x 10-6 M DES (Fig. 1BGo), as revealed by the presence of holes within the seminiferous cords. By D2, a large number of holes were visible. Similarly, by D2, degenerative germ cells were also often visible in the cords. By D3, DES had altered the architecture of the cords, and degenerating cells were common in all histological sections (Fig. 1DGo).



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FIG. 1. Effect of 4 x 10-6 M DES on the organization of the seminiferous cords in 14.5-day-old rat fetuses. One testis was cultured in control medium for 1 (A) or 3 days (C), and the other testis from the same fetus was incubated in medium containing 4 x 10-6 M DES for 1 (B) or 3 days (D). When the fetal testis was cultured in control medium, the integrity of seminiferous cords architecture was maintained (A and C), and numerous gonocytes were present (->). After just 1 day in the presence of 4 x 10-6 M DES (B), some gonocytes disappeared, as revealed by the holes (triangle marker) within the seminiferous cords. Note that the boundaries of the seminiferous cords indicated by the dashed lines were much more difficult to see after 3 days in culture with 4 x 10-6 M DES, and degenerative cells were frequently seen in all histological sections.

 
Effect of E2 and DES on the Number of Gonocytes
When explanted 14.5-d.p.c. rat fetal testes were cultured for 3 days in the presence of 4 x 10-6 M E2, the total number of gonocytes decreased by about one third (Fig. 2Go). When the testes were cultured in 4 x 10-6 M DES, the number of gonocytes decreased by one third after just one day (Fig. 3AGo). This decrease was concentration- and time-dependent: -31% by D1, -60% by D2, and -80% by D3. In the presence of 4 x 10-8 M DES, the number of gonocytes (–16%) was significantly reduced on D3. When 4 x 10-6 M ICI 182.780 (anti-estrogen) was added to the culture 1 h prior to stimulation with 4 x 10-8 M DES, the inhibitory effect of DES was abolished (Fig. 3AGo). ICI 182.780 (4 x 10-6 M) had no effect on the number of gonocytes on D3 when it was used alone (data not shown). We could not assess the antagonizing action of ICI on the effects of 4 x 10-6 M DES, due to its cellular toxicity at 4 x 10-4 M concentration, which would have been required to perform such an experiment. At 4 x 10-10 M, DES had no significant effect on the gonocyte population on D3 (Fig. 3AGo). It is noteworthy that the mean diameter (D) of the gonocyte nuclei was not altered by any of the treatments (D = 10.03 ± 0.03 µm; n = 54).



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FIG. 2. Effect of E2 on the total number of gonocytes in the 14.5-day-old rat fetal testes cultured for 3 days. One testis from each fetus was cultured in control medium and the other in medium supplemented with 4 x 10-6 M E2. At the end of the culture period, gonocytes were counted on histological sections. Values are means ± SEM of seven animals (A). BrdU was added to the dishes for the last 3 h on the second day of culture and the percentage of BrdU-positive gonocytes was measured in at least 1000 cells (n = 4) (B). DNA fragmentation was revealed by the TUNEL method, and the percentage of TUNEL-positive gonocytes was measured in at least 1000 cells (n = 4) (C). The numbers in brackets indicate the percentage of decrease, relative to control. *p < 0.05, **p < 0.015, in the paired statistical comparison with the corresponding control values.

 


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FIG. 3. Effect of DES and the anti-estrogen, ICI 182.780, on the total number of gonocytes in 14.5-day-old rat fetal testes cultured for 1, 2, or 3 days (D1, D2, or D3). One testis from each fetus was cultured in control medium and the other in medium supplemented with 4 x 10-6 M (n = 4), 4 x 10-8 M (n = 11) or 4 x 10-10 M DES (n = 4), or with DES plus 4 x 10-6 M ICI 182.780 (n = 11). At the end of the culture period, gonocytes were counted on histological sections (A). BrdU was added to the dishes for the last 3 h on the second day of culture, and the percentage of BrdU-positive gonocytes was measured in at least 1000 cells (n = 4) (B). DNA fragmentation was revealed by the TUNEL method, and the percentage of TUNEL-positive gonocytes was measured in at least 1000 cells (n = 7) (C). The numbers in brackets indicate the percentage of decrease or increase, relative to control. *p < 0.05, **p < 0.015 in the paired statistical comparison with the corresponding control values.

 
Effect of E2 and DES on Gonocyte Proliferation and Apoptosis
The mitotic and apoptotic indexes of gonocytes in 14.5-d.p.c. testes were evaluated after 3 days of culture in the presence of E2 (Figs. 2BGo and 2CGo). The percentage of BrdU-labeled gonocytes was reduced by 18% with 4 x 10-6 M E2 (Fig. 2BGo). Furthermore, the number of TUNEL-positive gonocytes in E2-treated testes was unchanged (Fig. 2CGo).

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. 3BGo and 3CGo). The percentage of BrdU-labeled gonocytes had dropped by 22% with 4 x 10-6 M DES (Fig. 3BGo). Furthermore, the number of TUNEL-positive gonocytes in DES-treated testes was 226% of the control values (Fig. 3CGo).

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. 4AGo). 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. 4AGo).



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FIG. 4. Effect of DES and the anti-estrogen, ICI 182.780, on the number of Sertoli cells in 14.5-day-old rat fetal testes cultured for 1, 2, or 3 days (D1, D2, or D3). One testis from each fetus was cultured in control medium and the other in medium supplemented with 4 x 10-6 or 4 x 10-8 M DES, or with DES plus 4 x 10-6 M ICI 182.780. At the end of the culture period, Sertoli cells, which were immunolabeled with GATA-4, were counted on histological sections (A). BrdU was added to the cultures for the last 3 h, and the percentage of BrdU-positive Sertoli cells was measured in at least 1000 cells (B). All values are means ± SEM of four animals. The numbers in brackets indicate the percentage of decrease, relative to control. *p < 0.05 in the paired statistical comparison with the corresponding control values.

 
Effect of DES on Sertoli Cell Proliferation and Apoptosis
The rate of Sertoli cell apoptosis was not affected by DES (data not shown). However, on D2, Sertoli cell proliferation was inhibited by 20% by 4 x 10-6 M DES (Fig. 4BGo). The diameter of the Sertoli cell nuclei was not changed (D = 8.41±0.05 µm; n = 16) by any of the treatments.

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. 5Go).



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FIG. 5. Effect of DES on basal and FSH-stimulated cAMP secretion by the 14.5-d.p.c. fetal testes cultured for 3 days. Three testes from three fetuses were cultured in the same control medium and the others in a medium supplemented with 4 x 10-6 M DES. The media were replaced by media containing IBMX (1 mM) with (+ FSH) or without (– FSH) 200 mUI/ml hFSH for the last 3 h. The cAMP in the media was measured by RIA. Values are means ± SEM of seven animals without FSH and eight animals with FSH. The numbers in brackets indicate the percentage of decrease, relative to control. **p < 0.015 in the paired statistical comparison with the corresponding control values.

 
Effect of DES on the Number of Leydig Cells
Although 4 x 10-8 M DES had no effect on the number of Leydig cells by D3, 4 x 10-6 M DES reduced the number of Leydig cells by 26% (Fig. 6Go). The diameter of the Leydig cell nuclei was not changed (D = 7.71±0.05 µm; n = 16) by any of the treatments.



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FIG. 6. Effect of DES on the proliferation of Leydig cells in 14.5-day-old rat fetal testes. The testicular explants were cultured for 3 days. One testis from each fetus was cultured in control medium and the other in medium supplemented with 4 x 10-6 M or 4 x 10-8 M DES. At the end of the culture period, the total number of Leydig cells per testis was evaluated. The Leydig cells were identified by 3ßHSD immunohistochemistry. The numbers in brackets indicate the percentage of decrease, relative to control. All values are means ± SEM of four animals. *p < 0.05 in the paired statistical comparison with the corresponding control values.

 
Effect of DES and E2 on Leydig Cell Activity
Explanted 14.5-d.p.c. testes were cultured for 72 h, with or without E2 or DES, and basal testosterone production was measured by RIA. We also added LH (100 ng/ml) to the media for the last 3 h of culture and assayed the LH-stimulated production of testosterone. Incubation of the fetal testes with 4 x 10-6 M E2 inhibited basal and LH-induced testosterone production by 56% and 54%, respectively (Fig. 7Go). DES also inhibited basal testosterone production (37% in the presence of 4 x 10-8 M DES and 45 % in the presence of 4 x 10-6 M DES) (Fig. 8Go). Furthermore, LH-induced testosterone production was decreased by more than one third in the presence of 4 x 10-10 M to 4 x 10-6 M DES (Fig. 8Go). Interestingly, when 4 x 10-6 M ICI 182.780 was added to the culture 1 h before stimulation with 4 x 10-8 M DES, the inhibitory effect of DES was abolished (Fig. 8Go). The culture of fetal testes with 4 x 10-6 M ICI alone had no significant effect on the Leydig cell number or on basal or LH-induced testosterone production (data not shown).



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FIG. 7. Effects of E2 on in vitro basal and LH-stimulated testosterone secretion by 14.5-d.p.c. rat fetal testes cultured for 75 h. One testis from each fetus was cultured in the control medium and the other in medium containing 4 x 10-6 M E2. The media were changed every 24 h and were supplemented with 100 ng/ml oLH from 72 to 75 h. Testosterone was measured by RIA. Values are means ± SEM of eight animals. The numbers in brackets indicate the percentage of decrease, relative to control. ***p < 0.001 in the paired statistical comparison with the corresponding control values.

 


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FIG. 8. Effects of DES and the anti-estrogen, ICI 182.780, on in vitro basal and LH-stimulated testosterone secretion by 14.5-d.p.c. rat fetal testes cultured for 75 h. One testis from each fetus was cultured in the control medium and the other in medium with various concentrations of DES or with DES plus 4 x 10-6 M ICI 182.780. All media were changed every 24 h and were supplemented with 100 ng/ml LH from 72 to 75 h. Testosterone in the media was measured by RIA. Values are means ± SEM of six animals in experiment with 4 x 10-6 M DES, eight animals in experiments with 4 x 10-8 M DES, and seven animals in experiments with 4 x 10-10 M DES. The numbers in brackets indicate the percentage of decrease, relative to control. *p < 0.05, **p < 0.015, ***p < 0.001 in the paired statistical comparison with the corresponding control values.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main objective of this work was to study the direct effect of E2 and DES on the fetal testes in culture.

We used an organ culture system and previously validated methods (Habert et al., 1991Go; Lecerf et al., 1993Go; Livera et al., 2000Go, 2001Go; Olaso et al., 1998Go) 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-{alpha} and -ß (Kuiper et al., 1997Go), to 4 x 10-6 M, which alters the proliferation of purified gonocytes in culture (Li et al., 1997Go). 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, 1993Go), 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, 2001Go). 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)Go 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., 1991Go), TGFß (Olaso et al., 1998Go), and retinoids (Livera et al., 2000Go). 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., 1999Go). 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., 2000Go; van Pelt et al., 1999Go) and ER-{alpha} (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., 1997Go). This finding is contradictory with the findings obtained by Li et al. (1997)Go, 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., 1997Go).

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., 1998Go). 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., 1998Go), whereas Leydig cells express both ER-{alpha} (Fisher et al., 1997Go) and ER-ß (O’Donnell et al., 2001Go), as do gonocytes (ER-{alpha}: our unpublished results; ER-ß: O’Donnell et al., 2001Go). 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., 2000Go; Livera et al., 2001Go) 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., 1993Go; Rannikki et al., 1995Go). 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, 1993Go). 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., 1986Go). Our data are also consistent with the results of a study by Majdic et al. (1996)Go, 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., 1997Go; Saunders et al., 1998Go; van Pelt et al., 1999Go). 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., 2000Go). Leydig cells differentiate from mesenchymal cells and do not enter mitosis (Boulogne et al., 1999Go). 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., 1999Go). An in vivo study by Majdic et al. (1996)Go 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., 1999Go; Saez, 1994Go), 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.


    ACKNOWLEDGMENTS
 
The authors wish to thank Drs. E. Chambaz, G. Defaye (INSERM, Grenoble, France), and J. M. Saez (INSERM, Lyon, France) for donating the antibodies anti-3ßHSD and anti-cAMP. Our thanks also go to C. Pairault and Dr. R. Olaso (Université Paris 7, France) for helpful advice. J. Lassurguère holds a fellowship from the Région Bretagne, and G. Livera is a recipient of a fellowship from the Ministère de l’Éducation Nationale de la Recherche et de la Technologie.


    NOTES
 
This work was supported by INSERM, Université Paris 7, and the Ministère de l’Aménagement du Territoire et de l’Environnement, and by the EEC EDEN grant QLX4-CT-2002-00603.

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. Back


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