Phthalates Impair Germ Cell Development in the Human Fetal Testis in Vitro without Change in Testosterone Production

1Fetal life is a critical step in the development of male reproductive functions. Indeed, the two major functions of the testis, gametogenesis and steroidogenesis, take place during this period. In humans, testis formation begins by the migration of primordial germ cells (PGCs) from extraembryonic areas to the genital ridge during the fifth week of gestation. Sertoli cells then differentiate in the gonadal ridge and surround the germ cells to form the seminiferous cords between the sixth and seventh weeks. At this time, the PGCs are called gonocytes. In parallel, Leydig cells differentiate from mesen-chymal cells in the interstitial compartment. These steroidogenic cells are morphologically discernible at 8 weeks of gestation, whereas in organ culture, testosterone secretion is detected from 6 weeks. The appropriate onset of gametogenesis and steroido genesis is fundamental for the function of reproduction in the adult. Indeed, the number of germ cells formed during fetal life is essential for adult fertility. In mutant, germ-cell–deficient (gcd−/−) mice characterized by a reduced number of PGCs, as in mice lacking the POG (proliferation of germ cells) gene, the number of fetal germ cells is reduced and adult fertility is altered. In the same way, androgens and insulin-like factor 3 (Insl3) produced by fetal Leydig cells control the masculinization of the reproductive tract and genitalia.

2Several studies have described an increasing frequency of male reproductive dis orders in humans, such as a low sperm count and a resulting decline in fertility, increased incidence of testicular cancer, cryptorchidism, and hypospadias (reviewed by; ). It has been suggested that these alterations are symptoms of a single entity called testicular dysgenesis syndrome (TDS) (reviewed by; ). It is currently thought that TDS is probably caused by changes in the development of the fetal testis and may result from the effect of genetic and/or environmental factors. Thus, TDS could result from exposure to environmental chemicals, which have steadily increased in diversity and concentration in the environment and food. Several environmental chemicals are classed among the so-called endocrine disruptors. Many of them act on reproductive functions because of their estrogenic and/or antiandrogenic properties. In the present study we focused on the effects of phthalates (phthalic acid esters), which are industrial chemicals commonly found in many consumer products regularly used by humans, such as soap, shampoo, cosmetics, and hairspray. They are also used in flexible plastics, such as food and beverage packaging, children’s toys, and biomedical equipment (e.g., blood transfusion bags). Di-2-ethylhexyl phthalate (DEHP) is one of the most abundant phthalates produced. demonstrated that phthalates, when administered orally to humans and rodents, are rapidly hydrolyzed by esterases in the gut and other tissues to produce the corresponding active monoesters. For example, DEHP is metabolized to its monoester metabolite, mono-2-ethylhexyl phthalate (MEHP), which is a recognized active testicular toxicant. Phthalates are not covalently bound to plastic products and therefore may leak out to contaminate blood or food products and can be ingested. In an epidemiologic study, 75% of the 289 human subjects tested were positive for the presence of four different types of phthalates in their urine samples. In rodents, both in vivo and in vitro approaches have been used to determine the effects on testicular functions of exposure to phthalates (reviewed by ). Several studies have shown that fetal exposure to di(n-butyl) phthalate (DBP) induced by gavage of pregnant rats induces TDS-like effects.

3However, despite the growing body of literature on phthalate reproductive toxicity and data demonstrating extensive human exposure, very few studies have examined the effects of these chemicals on human reproductive development. Recently, an inverse correlation has been shown between the maternal urinary phthalate concentration at the end of pregnancy and the anogenital distance at birth. In the same way, a dose-dependent association between phthalates in breast milk and levels of reproductive hormones in boys at 3 months of age has also been reported. These findings are particularly important because they are the unique epidemiologic studies exhibiting negative effects of phthalates at environmental concentrations. Until now, no experimental study has succeeded in demonstrating a deleterious effect of phthalates on human testis functions or development.

4In this study, we focused on phthalate effects specifically on the testis. We used the organ culture system of human fetal testes that we developed previously, coupled with morphologic, functional, and molecular methods, to analyze the effects of MEHP on the development of testicular somatic and germ cells during the first trimester of pregnancy (7–12 weeks of gestation). This early developmental period of the testis has been shown to be a critical window for the determination of the reproductive tract.

5Human fetal testes were obtained from pregnant women referred to the Department of Obstetrics and Gynecology at the Antoine Béclère Hospital for legally induced abortion in the first trimester of pregnancy, that is, from the seventh until the twelfth week of gestation, as previously described. None of the terminations was for reasons of fetal abnormality, and all fetuses appeared morphologically normal. The sex of the fetus was determined by the morphology of the gonads, and the fetal age was evaluated by measuring the length of limbs and feet. The fetuses were dissected under a binocular microscope; testes were removed aseptically and immediately explanted in vitro. We found testes within the abortive material in only 12% of cases. The Antoine Béclère Hospital Ethics Committee approved this study.

6We cultured testes on Millicell-CM Biopore membranes (pore size, 0.4 μm; Millipore, Billerica, MA, USA) as previously described. We used phenol red–free Dulbecco’s modified Eagle’s medium/ Ham F12 (1:1) (Gibco, Grand Island, NY, USA) supplemented with 80 μg/mL gentamicin (Sigma, St. Louis, MO, USA) and devoid of hormones, growth factors, and serum. We obtained MEHP from TCI Europe (Antwerp, Belgium).

7Each human testis was cut into small pieces, and all pieces from the same testis were placed on Millicell membranes floating on 320 μL culture medium in tissue culture dishes. Tissues were cultured for 4 days at 37°C in a humidified atmosphere containing 95% air/5% carbon dioxide, and the medium was changed every 24 hr. We measured the responses to MEHP (10−6, 10−5, and 10−4 M) by comparing one testis cultured in medium containing the tested factor with the other testis from the same fetus cultured in control medium. We added luteinizing hormone (LH; 100 ng/mL) from human pituitary (≥ 5,000 IU/mg; Sigma) or ketoconazole (KTZ; 4 μM; Sigma) every 24 hr to the culture medium. Bromodeoxyuridine (BrdU 30 μg/mL; Amersham Biosciences, Little Chalfont, UK) was added during the last 3 hr of culture for the measurement of proliferating index. At the end of the culture period, explants were frozen in RLT buffer (Qiagen, Valencia, CA, USA) at −20°C for RNA analyses, or dry frozen with liquid nitrogen for protein analyses. For cellular analyses, the explants were fixed for 2 hr in Bouin’s fluid, embedded in paraffin, and cut into 5-μm sections.

8We mounted serial sections on slides, removed the paraffin, and rehydrated the sections. We then carried out immunohistochemical assays for anti-Müllerian hormone (AMH) as previously described using an anti-AMH polyclonal antibody (1:2,000; generously provided by N. Di Clemente, INSERM U782, Clamart, France). We visualized peroxidase activity using 3,3′-diaminobenzidine as substrate. Germ cells were identified as AMH-negative cells within the seminiferous cords, whereas Sertoli cells were AMH positive. Counting was performed as previously described and validated for rodents and humans. Briefly, we counted germ cells in 1 of 10 sections for the 7-week-old human fetuses and 1 of 20 sections for later stages, but never fewer than 10 sections equidistantly distributed along the pieces of testis. We performed all counts using Histolab analysis software (Microvision Instruments, Evry, France). We counted all germ cells on the section and multiplied the sum of the values obtained for the observed sections of one testis by 10 or 20, respectively, to obtain a crude count of germ cells per testis. We then applied the Abercrombie formula, which uses the average measured diameter of the germ cell nuclei and the thickness of sections to correct for any double counting due to single cells appearing in two successive sections. All counts were carried out in a blind fashion.

9Because caspase-3 is involved in most of the apoptotic pathways, we used immunodetection of caspase-3 to quantify the rate of apoptosis as previously described. We mounted six sections on a single slide and heated the slide for 30 min in a permeabilization solution (0.05 M Tris, pH 10.6). The procedure was then the same as for detection of AMH, except that the primary antibody was anti-cleaved caspase-3, (1:50; Cell Signaling Technology, Beverly, MA, USA). Stained and unstained germ and Sertoli cells were counted in all six sections. For all immunohistochemical staining, we used tissue sections prepared without the primary antibody as negative controls.

10We labeled testes with BrdU (labeling reagent diluted 1:100 according to the instructions of the cell proliferation kit; Amersham Biosciences) during the last 3 hr of culture. BrdU incorporation into proliferating cells was detected by immunocytochemistry according to the manufacturer’s recommendations, as previously described. The BrdU incorporation index was obtained by a blind counting of stained and unstained germ or Sertoli cell nuclei in all sections.

11We measured the testosterone secreted into the medium in duplicate by radioimmunoassay as previously described. No extraction or chromatography was performed because 17β-hydroxy-5α-androstan-3-one (DHT), the only steroid that significantly cross-reacts with testosterone (64%), is secreted in minute amounts by the fetal testis.

12We performed RNA expression in the fetal testis by reverse transcriptase (RT) with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Courtabeuf, France), followed by real-time polymerase chain reaction (PCR) as previously described. Primers and probes used were designed by Applied Biosystems: β-Actin primer, GenBank accession no. NM_001101.2; AMH, Probe accession no. Hs00174915_ m1; Insl3, Hs01394273_m1; P450c17 (cytochrome P450 c17a), Hs00164375_m1; P450scc (cytochrome P450 11A1), Hs00167984_m1; RPLPO (large ribosomal protein PO), NM_053275.3; StAR (steroidogenic acute regulatory protein), Hs00264912_m1; and Wt1 (Wilms tumor 1), Hs01103749_m1. Reactions were carried out and fluorescence was detected using an ABI Prism 7000 apparatus (Applied Biosystems). Each sample was run in triplicate, and negative controls were run for every primer/probe combination. The measured amount of each cDNA was normalized using β-actin and RPLPO or Wt1 for AMH.

13One testis was lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na4O7P2, 1 mM β-glycerol phosphate, 1 mM phenylmethylsul-fonyl fluoride, 1 mM Na3VO4, and 1 μg/mL leupeptin. Protein in total cell lysates (5 μg) was resolved by sodium dodecyl sulfate poly-acrylamide gel electrophoresis, electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham Biosciences), and probed with antibodies for AMH (same as for immunohistochemistry) and β-actin (Sigma). We used Cy5-coupled anti-rabbit and Cy3-coupled anti-mouse secondary antibodies (Amersham Biosciences), and the blot was revealed under fluorescence in a Typhoon 9400 scanner (Amersham Biosciences). We quantified the bands by the volumetric method with the software ImageQuant (Molecular Dynamics, Sunnyvale, CA, USA).

14All values are expressed as mean ± SEM. For all mRNA expression analysis and studies on proliferation or apoptosis, we evaluated the significance of the differences between mean values for the treated and untreated testes from the same fetus using Wilcoxon’s nonparametric paired test (for small samples). For total germ cell number counting, we used Student’s paired t-test because of the high variability in the number of germ cell between ages. Concerning testosterone secretion analysis, we used one-way analysis of variance (ANOVA) to assess the significance of the differences for secretion evolution between control and treated testes during the 3 days of culture.

15We cultured testes from fetuses at 7–12 weeks of development with or without 10−6, 10−5, or 10−4 M MEHP for 4 days. Daily testosterone production was unaffected by the addition of MEHP to the medium (Figure 1A). To check the lack of effect of MEHP, we analyzed the mRNA expression of various enzymes involved in steroidogenesis. MEHP treatment did not affect the mRNA expression of P450c17, P450scc, or StAR (Figure 1B). MEHP did not modify mRNA expression of Insl3 produced by fetal Leydig cells, which is known to be involved in testicular descent (Figure 1C).

16To assay the ability of testosterone secretion to be modulated in our organotypic culture system, we performed cultures with LH for testosterone stimulation and with KTZ for testosterone inhibition. With 100 ng/mL LH, the relative testosterone secretion was increased 5-fold at day 3 (Figure 2B). On the other hand, treatment with 4 μM KTZ (a cytochrome P450 inhibitor), which we have determined to be a nontoxic concentration for the testis (Figure 2A), induced very strong inhibition of testosterone production from day 2. Using one-way ANOVA, the change in testosterone secretion with both LH and KTZ treatments differed significantly from their respective controls. These results strengthen the validity of testosterone measurement in this model.

17To investigate the effect of MEHP on stimulated testosterone secretion, testes were cultured in the presence of LH (100 ng/mL) with or without 10−4 M MEHP. Relative LH-stimulated testosterone production was unaffected by the addition of MEHP to the medium (Figure 3).

18We studied the ratio of proliferative (BrdU positive; Figure 4A) and apoptotic (cleaved caspase-3 positive; Figure 4B) Sertoli cells after MEHP treatment for 3 days and observed that MEHP had no significant effect on these two activities.

19We also analyzed the effect of MEHP on AMH expression by real-time RT-PCR (Figure 5A) and by fluorescent Western blotting (Figure 5B). Regardless of the housekeeping gene (β-actin or RPLPO) or specific Sertoli cell marker (Wt1, which is not significantly different in control and treated samples if standardized to β-actin), MEHP significantly decreased the mRNA level of AMH. However, the level of AMH protein standardized to β-actin was not modified by MEHP treatment.

20Addition of 10−6 M, 10−5 M, or 10−4 M MEHP for 3 days had no effect on the organization of the testis at the end of the culture (data not shown). Interestingly, regardless of the age of the fetus at explantation (from 7 to 12 gestational weeks), the higher dose of MEHP (10−4 M) significantly reduced the number of germ cells. Therefore, we expressed the results as a percentage of control and pooled the results from different ages (Figure 6A). However, the 10−6 M concentration had no effect.

21Treatment with 10−4 M MEHP significantly increased the number of cleaved caspase-3–positive germ cells (Figure 6C,D) without altering their proliferation (Figure 6B). Treatment with 10−5 M MEHP increased, but not significantly, the number of cleaved caspase-3–positive germ cells (3.4% in the treated vs. 2% in the control testes) (Figure 6D).

22In this study, we investigated the effect of one metabolite of phthalate ester, MEHP, on the development of human fetal testes, using our previously developed and validated organ culture system. In this organ culture system, the testicular architecture and intercellular communications are preserved enough to allow the development of the main fetal testicular cell types in vitro, without any added factor.

23This approach allowed us to present here the first experimental demonstration that phthalates impair the development of the male fetal germ cell lineage in the human species. After 3 days of treatment, MEHP reduced by 40% the number of germ cells in cultured human fetal testis. This effect was due to a large increase in their apoptosis without modification of their proliferation. A negative effect of phthalates on gonocyte number has also been reported in rodents both in vivo, after gavage, and in vitro, in organ culture (Chauvigné F, Menuet A, Chagnon M-C, Lesné L, Jegou B, unpublished data; Lehraiki A, Szenker J, Habert R, Levacher C, unpublished data; ). It is interesting that, in rodents, the androgen pathway does not seem to be involved in germ cell number, because phthalates are distinct from flutamide in their ability to induce PGC degeneration.

24Phthalates induce the appearance of multi-nucleated gonocytes in rodents. In the present study, we observed no multinucleated gonocytes in response to MEHP treatment. This may be due to a species characteristic; appearance of multinucleated gonocytes in rodents depends on the age of the fetus. For example, observed multinucleated gonocytes after DBP gavage only from day 19.5 postconception. Thus, the sensitive window in humans may occur later than the period studied here.

25Phthalates are known as Sertoli cell toxicants in rodents. Some studies have reported a decrease in Sertoli cell number or proliferation, interference with cytoskeleton, and decrease in the expression of Sertoli cell markers (AMH, GATA4, inhibin) and of follicle-stimulating hormone–stimulated cAMP production. In this study, we observed that MEHP did not affect the proliferation or apoptosis rate of Sertoli cells. On the other hand, MEHP decreased the mRNA expression of AMH whether we normalized relative expression to ubiquitous expression or to the Sertoli gene. Surprisingly, however, the intracellular AMH protein level was not modified by MEHP treatment when analyzed by Western blot. To explain this discrepancy, we can hypothesize that MEHP affects RNA expression earlier than it does protein expression.

26In the rat, it is currently thought that phthalates act mainly on fetal Leydig cells. Thus, the main effects of phthalates are the suppression of testosterone production, an abnormal Leydig cell aggregation, and the presence of intratubular Leydig cells. Jegou and colleagues (Chauvigné F, Menuet A, Chagnon M-C, Lesné L, Jegou B, unpublished data) have also observed a similar decrease in testosterone production in organ culture of rat fetal testis, and we confirmed this result (data not shown). Nevertheless, have shown that MBP (monobutyl phthalate) reduces human chorionic gonadotropin–stimulated but not basal steroidogenesis in rat fetal testis explants. In the present study, we observed that MEHP changes neither the basal nor the LH-stimulated production of testosterone by the human fetal testis in culture. This was confirmed by the absence of modification of the mRNA levels of steroidogenic enzymes after MEHP treatment. also found no in vitro effect of phthalates (MBP and DBP) on steroidogenesis of human fetal testis explants recovered during the second (15–20 weeks) trimester of pregnancy. So, steroidogenesis of the human fetus during the first and second trimesters seems not to be sensitive to phthalates. This period in humans corresponds to the window studied in the rat model. It is interesting that, in mice, MEHP stimulates testosterone production both in the fetal testis (; Lehraiki A, Szenker J, Habert R, Levacher C, unpublished data) and in Leydig tumor cells. This lack of effect on testosterone production in humans suggests differences between species.

27The results presented here contrast with epidemiologic data in humans. We can formulate two hypotheses to explain this apparent discrepancy: first, the studies differ in phthalate concentration, nature, and duration of exposure. We chose to investigate the effect of phthalate concentrations ranging from 10−6 M to 10−4 M. In a prospective study on cryptorchidism carried out on the phthalate monoester contamination of human breast milk, found many phthalate monoesters over a large concentration range, from 1.5 to 1,410 μg/L, which corresponds to about 10−9 M to 10−6 M for MEHP. In our study, we observed no effect at the lower concentration, either on steroidogenesis or on gametogenesis. However, the mother and thus the fetus are exposed to a combination of multiple phthalates, which could explain the need for a greater dose of MEHP to show a potent effect in vitro. Second, in our study we focused on the effect of MEHP specifically on testis, so we cannot rule out that the observed effects in epidemiologic studies (cryptorchidism and anogenital distance) are due to a direct effect of phthalates on the reproductive tract.

28In the human fetus, intraabdominal testicular descent to the inner inguinal ring is initiated at about 10–14 weeks of gestation. A role for Insl3 secreted by differentiated Leydig cells, in development of the gubernaculum and this first phase of testicular descent, has emerged after analysis of mice genetically modified for Insl3 expression. Moreover, underdeveloped gubernaculum and reduced Insl3 expression have been observed after fetal exposure to several different phthalates in male rats. We observed no effect of 10−4 M MEHP (highest concentration) on the Insl3 mRNA in human testis culture, even though our study took place during the setup of the testicular descent. have recently demonstrated that MEHP represses Insl3 transcription by antagonizing testosterone action in Leydig cells. Thus, the absence of effect on Insl3 expression observed here can be explained by the lack of effect of MEHP on testosterone production in our model. Nevertheless, androgen receptor antagonists seem to have no effect on Insl3 expression.

29In conclusion, this is the first experimental demonstration that phthalates, a family of compounds known as endocrine disruptors, widely distributed in the environment, are able to alter the development of male germ cell lineage in humans. This effect is not mediated by a decrease in the testosterone produced by the Leydig cells, which is unchanged. Furthermore, this study shows the efficiency of our organ culture system in investigating the effects and mechanisms of action of environmental disruptors on the development of the human fetal testis. Lastly, our work provides important insight into the potential role of exposure to environmental pollutants during fetal testicular development and their potential deleterious effects on male fertility in adulthood.