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Testis |
School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gamete biology, Sertoli cells, testis, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Phthalates are not covalently bound to the plastic products, and therefore, they may leach out to contaminate blood or food products and can be ingested. The average daily exposure to DEHP for the general population is approximately 3–30 µg/kg daily [3]. It is also important to note that 75% of 289 human subjects tested were positive for four different types of phthalates in their urine samples [4]. Of even more concern is the finding that women of reproductive age have a significantly higher level of phthalates than other sex and age groups due to their use of phthalates that may originate from products such as perfume, lotion, hairspray, and nail polish [4]. Moreover, clinical exposure, such as exposure to DEHP from blood transfusions, could be as high as one to two orders of magnitude over the general exposure level. In fact, leaching of DEHP from blood transfusion bags leads to levels of DEHP in blood ranging from 10–650 µg/ml [3]. For critically ill infants with a smaller body size than adults, it is likely that the exposure levels can be as much as three orders of magnitude higher than the general exposure level during the developmentally sensitive period [3]. Therefore, these data raise a significant concern about the potential health risks to humans, especially to developing embryos and neonates.
Studies have demonstrated that exposure of rodents to DEHP or MEHP can impair neonatal and adult testis function [5, 6]. Testicular degeneration is characterized by Sertoli cell vacuolation, shedding of spermatocytes and spermatids, and ultimately decreased sperm production [1]. Treatment of pregnant rats with DEHP or di(n-butyl) phthalate (DBP) during gestation resulted in decreased anogenital distance, undescended testes, degeneration of seminiferous epithelium, interstitial cell hyperplasia, and decreased testis weight in the male offspring [7–9]. In addition, administration of DEHP or MEHP by gavage in neonatal rats altered morphology of gonocytes and impaired Sertoli cell proliferation [5]. Experiments using cocultures of germ cells and Sertoli cells showed that MEHP induced detachment of germ cells from the underlying Sertoli cell monolayer [10].
Even though experiments have been conducted in vivo to probe for the effect of in utero exposure of phthalates on the testis [8, 9], the direct effects of MEHP on sexual differentiation of indifferent gonad to the testis and subsequent development of the fetal testis remain largely unexplored. Sexual differentiation of the testis is a tightly regulated process that involves differentiation of Sertoli cells directed by Sry, the sex-determining gene on the Y chromosome expressed in pre-Sertoli cells, and subsequent migration of mesenchymal cells from mesonephros to the adjacent gonad forming seminiferous cords around Embryonic Day 13.5 (E13.5) in rats [11]. Sertoli cells aggregate and organize primordial germ cells inside the cords, as flattened peritubular myoid cells of mesonephric origin form the outer cell layer of the seminiferous cords encompassing both Sertoli cells and germ cells [12, 13]. Once the primordial germ cells are enclosed inside the testicular cords, they change morphologically and differentiate into gonocytes [14]. Gonocytes become mitotically quiescent until Postnatal Day 3 (P3), when they resume mitosis and migrate from their central position in the seminiferous tubules to the basement membrane [15, 16]. Sertoli cells, on the other hand, continue proliferation in fetal and neonatal stages, slow proliferation by 15 days of age, and stop proliferation around 18 days after birth [17]. Therefore, the final size of the Sertoli cell population is determined during the fetal to pubertal period [17, 18].
In this study, we used rat testicular organ cultures to assess the in vitro effect of MEHP on the testicular cord formation in E13 testis and the development of both gonocytes and Sertoli cells in fetal and neonatal testes. This in vitro organ culture method provides a unique advantage to study testis development while largely maintaining normal structural relationships among the different testicular cell types. It has been used to study neonatal germ cell development [15] and the effects of retinoic acid on the development of fetal and neonatal testes [19, 20]. Testes at three developmental stages were used: E13, when testicular cord formation and Sertoli cell differentiation begin; E18, when the Sertoli cells continue proliferating while gonocytes are mitotically quiescent; and P3, when gonocytes migrate to the basal side of seminiferous tubules and resume mitosis [15, 16, 21].
It has been demonstrated that mouse vasa homologue (MVH) protein is exclusively expressed in germ cells from E11.5 to the postmeiotic stage in both males and females [22]. Therefore, we used MVH immunohistochemistry to identify gonocytes in both fetal and neonatal testes. To assess the effect of MEHP on Sertoli cells, we used two Sertoli cell markers, mullerian inhibiting substance (MIS) and GATA-4. MIS, a member of the transforming growth factor ß family, is expressed exclusively in Sertoli cells in the mouse beginning between E11.5 and E12.5 until shortly after birth [23]. GATA-4, a member of the zinc finger family of transcriptional factors, is expressed in Sertoli cells at a high level during fetal and early postnatal development [24, 25] and has been shown to be involved in testis differentiation in mice [26]. Thus, both GATA-4 and MIS are widely used as functional markers for Sertoli cell differentiation [27, 28].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnant, female Sprague-Dawley rats were obtained from Charles River Laboratories (Hollister, CA). Plug date was considered E0. Animal experimentation was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the highest standards of humane animal care as outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Organ Cultures
E13 gonads with the mesonephroi were cultured as previously described [19]. Briefly, tissues were cultured on Millicell CM filters (Millipore, Bedford, MA) floating on the surface of CMRL 1066 medium (Life Technologies, Rockville, MD), supplemented with penicillin-streptomycin, insulin (10 µg/ml), and transferrin (10 µg/ml). One gonad was cultured in medium containing MEHP (50, 100, or 200 µM), and the other gonad from the same fetus was cultured in medium containing an equivalent volume of dimethyl sulfoxide (solvent for MEHP) as a control. MEHP (
99% pure) was generously synthesized by Dr. Jan Wahlstrom in the laboratory of Dr. Jeffrey Jones (Department of Chemistry, Washington State University). The organs were maintained for 3 days in culture, at which time testicular cords were well developed in the controls. The culture medium was changed every day. The data were obtained from testis organ cultures from at least three gestating female rats. In addition, the data were obtained from at least four male embryos from each gestating female rat. To determine the sex of E13 embryos, polymerase chain reaction analysis for Sry (sex-determining region, Y chromosome) was conducted on tail genomic DNA isolated from E13 embryos [19].
For organ cultures of testis from E18 embryos and P3 rats, testes were cut into halves for E18 and eight pieces for P3 testes. All the pieces from the same testis were cultured on a Millicell filter floating on 1 ml of CMRL 1066 medium (Gibco BRL, Grand Island, NY), supplemented with penicillin-streptomycin, and incubated at 37°C in an atmosphere of 5% CO2 for 3 days. The culture medium was changed every day. At the end of the culture, tissues were fixed for 1 h at room temperature in Bouin solution, embedded in paraffin, and cut into 5-µm sections. The data were obtained from testis organ cultures for E18 and P3 from at least three gestating, female rats. In addition, the data were obtained from at least four male embryos or pups from each gestating female rat.
Gonocyte Counting
Changes in the number of gonocytes were analyzed by counting the gonocyte nuclei in sections stained with hematoxylin-eosin. Gonocytes were easily identified by their relatively large, round, and lightly stained nuclei containing one to several globular nucleoli [29, 30]. The identity of gonocytes was confirmed using an antibody to MVH. Counts were made using an ocular grid (area, 56 406 µm2) at a final magnification of 200x. Gonocytes in 3 grids for E13 testis, 10 grids for E18 testis, and 20 grids for P3 testis were counted from three testicular sections, which were at least 20-µm intervals apart to avoid counting the same cell twice.
Immunohistochemistry
Immunohistochemistry was performed as described previously [31]. For detection of MIS, GATA-4, and MVH, the sections were incubated with goat polyclonal antibodies (MIS, 1:300; GATA-4, 1:150; Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit polyclonal antibody against MVH (1:1000) [22] in a humidified chamber overnight at 4°C. Sections were washed with PBS and treated with biotinylated rabbit anti-goat secondary antibody (Zymed Laboratories, South San Francisco, CA) for MIS and GATA-4 or biotinylated goat anti-rabbit secondary antibody for MVH (1:300, Vector Laboratories, Burlingame, CA), followed by incubation with peroxidase-conjugated streptavidin and substrate-Chromagen mixture containing aminoethyl carbazole from Zymed Laboratories. As negative controls, serial sections were incubated with nonimmune serum or primary antibody preabsorbed with a 50-fold excess of synthetic immunizing peptide (Santa Cruz Biotechnology). Immunohistochemistry was performed on the testis sections from at least three embryos or P3 rats.
Proliferation Analysis
5-Bromo-2'-deoxyuridine (BrdU) at 1:100 dilution from Zymed Laboratories was added to the culture medium 3 h before the completion of the culture. BrdU immunohistochemistry on tissue sections was performed following the protocols provided by the supplier (Zymed Laboratories). Briefly, randomly chosen sections were treated with 3% H2O2 in methanol to quench endogenous peroxidases and immersed in 1 N HCl for 30 min to denature the genomic DNA. After rinsing, the sections were treated with blocking solution and incubated for 1 h with biotin-conjugated anti-BrdU monoclonal antibody (Zymed Laboratories). Subsequently, the sections were treated with streptavidin peroxidase complex solution for 10 min and visualized by a reaction with substrate-Chromagen mixture (Zymed Laboratories). All sections were lightly counterstained with hematoxylin for clear assignment of labeled nuclei as Sertoli cells or gonocytes and for obtaining the number of unlabeled cells. At least 900 Sertoli cell or 200 gonocyte nuclei were counted for three nonconsecutive sections from every treatment group per experiment. The BrdU labeling index (percentage of BrdU-labeled cells) was calculated from the number of BrdU-labeled Sertoli cells or gonocytes divided by the total number of Sertoli cells or gonocytes and multiplied by 100.
Assay for Apoptosis
TUNEL assay was performed as described by the manufacturer of the Apoptosis Detection System (Promega Biotech Corporation, Madison, WI). This assay detects fragmented DNA in apoptotic cells by catalytic incorporation of fluorescein-12-dUTP at the 3'-OH ends of DNA using the terminal deoxynucleotidyl transferase enzyme. The fluorescein-12-dUTP labeled DNA can then be visualized directly under a fluorescence microscope. To quantify the relative differences in the number of apoptotic cells between the control and treated testes, the percentage of 80 seminiferous tubules containing three or more TUNEL-positive cells were determined for three sections per treatment.
Statistical Analysis
All values are means ± SDs. Statistical analysis was performed using one-way ANOVA followed by pairwise comparison of the means at P = 0.05 (Tukey-Kramer test, Minitab 10 Xtra, Minitab Inc., State College, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of MEHP on testicular cord formation were assessed using E13 organ cultures. Both the control gonads and the gonads treated with variable doses of MEHP (50, 100, and 200 µM) formed testicular cords after 3 days of culture (Fig. 1, A–D). Further analysis of the testicular sections from these organs, which were stained with hematoxylin-eosin, showed that the testicular cords were well developed and the types of cells present in the testicular cords were normal looking both in control testes and MEHP-treated testes (Fig. 1, E–H). To determine the potential cellular target for phthalates in the gonad undergoing testicular differentiation, the expression patterns of MIS, GATA-4, and MVH were examined. MIS was found at high levels specifically within Sertoli cells lining the testicular cords, and MIS expression in the E13 testes treated with MEHP remained similar to that of the controls (Fig. 2, A, B, E, and F). Likewise, GATA-4 expression was unchanged (data not shown). In germ cells, MVH in the control testes was similar to the treated testes (Fig. 2, C, D, G, and H). In addition, the number of gonocytes in the E13 testes was not changed significantly by MEHP treatment, as determined by counting the nuclei of gonocytes on the sections stained with hematoxylin-eosin (data not shown). This was confirmed by comparison of the number of MVH-positive cells in MEHP-treated E13 testes with that of the control testes.
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MEHP Decreases the Levels of Both MIS and GATA-4 in Sertoli Cells and Impairs Sertoli Cell Proliferation Without Changing the Number of Gonocytes in E18 Testes
To determine the potential cellular target of phthalates in E18 testes, the expression levels of Sertoli cell and germ cell markers and the proliferation index for Sertoli cells were determined. Additionally, the number of gonocytes was counted. Both MIS and GATA-4 protein levels in Sertoli cells were similar to controls in the E18 testes treated with 50 µM MEHP (Fig. 3). In contrast, the levels of both proteins decreased markedly in the organ cultures of E18 testes treated with MEHP at 100 or 200 µM when compared with those in the control testes (Fig. 3). Correspondingly, the BrdU labeling index of Sertoli cells in the testes treated with 100 or 200 µM decreased significantly compared with that in the controls, whereas a slight, but not significant, decrease in the BrdU labeling index of Sertoli cells was observed in the testes treated with 50 µM MEHP (Fig. 4). The organization of gonocytes and Sertoli cells in the seminiferous cords was not altered by MEHP treatment (Fig. 5, A–D). Similarly, the expression level of MVH in the gonocytes of the E18 testes did not change after MEHP treatment (Fig. 5, E–H). In addition, the number of gonocytes in the E18 testes remained similar whether the testes were treated or untreated with MEHP for 3 days (data not shown).
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MEHP Decreases the Levels of MIS and GATA-4 in Sertoli Cells and Impairs Sertoli Cell Proliferation in P3 Testes
The steady-state expression levels of MIS and GATA-4 were also used to assess the effect of MEHP on Sertoli cells in the organ cultures of P3 testes. Both MIS (Fig. 6, A–D) and GATA-4 (Fig. 6, E and F) protein levels in Sertoli cells dramatically decreased in the organ cultures of P3 testes treated with MEHP in a dose-dependent manner compared with those in the control testes. In addition, MEHP at 100 or 200 µM significantly decreased the BrdU labeling index of Sertoli cells (Figs. 7 and 8). The decrease in the BrdU labeling index of Sertoli cells observed in the testes treated with 50 µM MEHP was not significant (Fig. 8).
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MEHP Decreases the Number of Gonocytes by Increasing Apoptosis in P3 Testes
The effect of MEHP on the gonocytes in P3 testis after 3 days of culture was assessed using both hematoxylin-eosin staining (Fig. 9, A–D) and immunohistochemistry for MVH (Fig. 9, E–H). After 3 days of culture, the integrity of the seminiferous cord structure was maintained. Some germ cells were obviously near the basement membrane of the seminiferous tubules, whereas others were localized to the lumen of the seminiferous tubules in the control P3 testes (Fig. 9, A). In comparison, both Sertoli and germ cells in the seminiferous tubules of P3 testes treated with 100 or 200 µM MEHP (Fig. 9, C, D, G, and H) were disorganized. In addition, there were some abnormally large and irregularly shaped gonocytes observed mostly in the lumen but also near the basement membrane of the seminiferous tubules. Furthermore, gonocyte counting revealed that the number of gonocytes significantly diminished in the P3 testis after treatment with MEHP at 100 or 200 µM compared with that in the control testes (Fig. 10).
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To determine whether MEHP decreased the number of gonocytes by decreasing their proliferation or increasing apoptosis, the effect of MEHP at concentrations of 100 and 200 µM, was evaluated. MEHP at concentrations of 100 and 200 µM did not alter the BrdU labeling index of gonocytes in P3 testes significantly after 3 days of culture (data not shown). However, compared with the few TUNEL-positive cells observed in the control testes, there was an increase in the TUNEL-positive cells in the MEHP-treated testes (Fig. 11). Most of the apoptotic cells appeared to be gonocytes according to their morphology and location in the lumen of the seminiferous tubules. When the percentage of seminiferous tubules containing three or more TUNEL-positive gonocytes per seminiferous tubule was determined, there was a significant increase in apoptotic tubules in the testes treated with MEHP at concentrations of 100 and 200 µM (Fig. 12).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Formation of seminiferous cords at E13.5 is the first defined morphological change in the testis during sexual differentiation. Interestingly, MEHP had no effect on testicular cord formation even when E13 testis with mesonephros was treated with a high dose of MEHP (200 µM). The organization of Sertoli cells and gonocytes inside the seminiferous cords and flattened peritubular myoid cells surrounding both Sertoli and gonocytes was normal. In addition, the differentiated function of Sertoli cells was apparently not altered as demonstrated by the expression of MIS, a functional marker of Sertoli cell differentiation, in the testes after MEHP exposure. Moreover, the number of gonocytes in the MEHP-treated testes remained similar to that in the control testes. Our results are compatible with an in vivo study, which showed that exposure of DBP, one of the phthalate diesters, in rats during gestation had no effect on the morphology of E16 testis [8].
We also studied the effect of MEHP on the testis at a late fetal stage (E18) and neonatal stage (P3), when Sertoli cells remain proliferative [17, 18]. MEHP treatment decreased the proliferation of Sertoli cells in the testes from both E18 embryos and P3 animals. Although it has been presented previously that MEHP impaired Sertoli cell proliferation in neonatal testis [5], to our knowledge, this is the first report that demonstrates that MEHP decreases Sertoli cell proliferation in fetal testes. It has been demonstrated that the size of the Sertoli cell population in the adult testis is established during the late fetal and neonatal period [17, 18, 32], and a reduction in Sertoli cell numbers due to decreased Sertoli cell proliferation in neonatal rats can result in a decrease in spermatid numbers in adults [33]. This is because each Sertoli cell can only support a finite number of germ cells, and therefore, the number of Sertoli cells determines the spermatogenic capacity in adults. Our observation that MEHP impairs Sertoli cell proliferation in the testes at both late fetal and neonatal developmental stages is a significant finding, since it implies that exposure of both fetal and neonatal testes to MEHP may diminish the size of the Sertoli cell population in the adult testis.
Although it is well known that MEHP can induce testicular toxicity in rodents, the mechanism of this adverse effect has not been fully explained. Both in vivo and in vitro studies have suggested that Sertoli cells are the primary cellular targets of MEHP-induced testicular toxicity [1, 5, 10, 34]. Previously, MEHP has been shown to disturb FSH interaction with the FSH receptor [35] and decrease FSH-stimulated cAMP accumulation in Sertoli cell cultures [36]. In the present study, we demonstrate that the expression levels of Sertoli cell proteins, GATA-4 and MIS, which are widely used as functional markers of Sertoli cell differentiation, are decreased in the testes at both late fetal and neonatal stages. These results further suggest that Sertoli cells are indeed cellular targets of MEHP not only during the neonatal developmental period [5, 10] but also during the late fetal developmental period.
Furthermore, the present study demonstrates that MEHP treatment alters the morphology and the function of gonocytes that are known to resume mitosis in the P3 testes. In contrast, the gonocytes that are mitotically quiescent in late fetal testis (E18) were not affected by MEHP exposure. Thus, interestingly, MEHP appears to target mitotically active gonocytes. The abnormally large appearance of the gonocytes occasionally observed in the P3 testes treated with MEHP has been reported previously when MEHP was administered to neonatal rats by gavage [5]. It was suggested that these gonocytes might be destined to die [5]. Indeed, MEHP treatment decreased the number of gonocytes in the P3 organ cultures by increasing apoptosis of gonocytes. This effect was demonstrated to be dose dependent.
Around P3, gonocytes not only resume mitosis but also migrate toward the basement membrane of seminiferous tubules [15, 16] and differentiate into spermatogonial stem cells or type A1 spermatogonia [37]. These developmental processes for germ cells, which are highly organized and tightly regulated, are postulated to be critical to establish normal spermatogenesis in adults [14, 33]. It is possible that MEHP disturbs the migration of gonocytes in neonatal testes due to abnormal Sertoli-gonocyte physical interactions. Experiments using cocultures of germ cells and Sertoli cells indicate that MEHP can induce detachment of germ cells from the underlying Sertoli cell monolayer [10, 38]. At any rate, it is accepted that if gonocytes remain in the lumen and fail to migrate, they undergo apoptosis [16, 39]. Thus, it remains to be seen if MEHP disturbs the mitosis and/or the migration of gonocytes toward the basement membrane, resulting in apoptosis of gonocytes and a decrease in the number of germ cells.
MEHP is thought to cause apoptosis in germ cells by first injuring Sertoli cells in the adult testis [40, 41]. The current paradigm is that when Sertoli cells are dysfunctional and are not able to adequately nurture the development of germ cells, they transmit an apoptotic signal for germ cells to die. Previously, MEHP has been shown to up-regulate Fas ligand (FasL), which is secreted by Sertoli cells, to initiate apoptosis of Fas-positive germ cells in the adult testis [40–46]. Alternatively, MEHP has been shown to induce oxidative stress selectively in the germ cells and cause apoptosis of spermatocytes [47], indicating that MEHP could act directly on the germ cells. Whether MEHP causes apoptosis in the gonocytes of neonatal testes by directly injuring gonocytes or indirectly by first causing an injury to Sertoli cells requires further investigation.
Testicular dysgenesis syndrome (TDS) is considered a common developmental disorder caused by environmental toxins [48]. It is generally accepted that dysfunction of Sertoli cells or Leydig cells in fetal or early life underlies the risk of TDS in humans, which is characterized by poor semen quality, undescended testis, hypospadias, and testicular cancer [48, 49]. In addition, testicular cancer is believed to be associated with aberrant development of fetal gonocytes [50]. The mechanisms involved in causing TDS are unknown. A recent study describes a rat model for TDS with partially formed seminiferous cords, immature Sertoli cells, Leydig cell hyperplasia, and multinucleated gonocytes in dysgenetic areas after in utero exposure of the rat to DBP [51]. Investigators concluded that Sertoli cell development in fetal and perinatal life is abnormal in DBP-exposed animals, which could then lead to abnormal testicular changes that resemble human TDS [51]. Consistent with the results from the rat model, Sertoli cells of the fetal (E18) and neonatal (P3) testes in culture and gonocytes of the neonatal testes in culture were abnormal. These results suggest that abnormal Sertoli cell proliferation and differentiation and abnormal gonocyte apoptosis may indeed be the fetal and neonatal events responsible for phthalate eliciting similar characteristic disorders in animal models as human TDS.
In summary, we investigated the effect of MEHP on organ cultures of rat fetal and neonatal testes to understand MEHP toxicity on the testicular cells. We found that MEHP did not affect early steps of fetal testis formation during sexual differentiation. However, MEHP decreased the levels of MIS and GATA-4 in Sertoli cells and impaired proliferation of Sertoli cells in both E18 and P3 testes. Moreover, MEHP decreased gonocyte numbers by increasing apoptosis in a dose-dependent manner in P3 testes. These observations indicate that MEHP affects testis development during both fetal and neonatal periods, critical times for establishment of normal testicular cell numbers and function in adults.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kwan Hee Kim, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234. FAX: 509 335 1907; khkim{at}wsu.edu
Received: 1 May 2003.
First decision: 26 May 2003.
Accepted: 30 July 2003.
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REFERENCES |
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expression in the rat epididymus. Biol Reprod 1996 54:1111-1119[Abstract]This article has been cited by other articles:
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  M. Culty, R. Thuillier, W. Li, Y. Wang, D. B. Martinez-Arguelles, C. G. Benjamin, K. M. Triantafilou, B. R. Zirkin, and V. Papadopoulos In Utero Exposure to Di-(2-ethylhexyl) Phthalate Exerts Both Short-Term and Long-Lasting Suppressive Effects on Testosterone Production in the Rat Biol Reprod, June 1, 2008; 78(6): 1018 - 1028. [Abstract] [Full Text] [PDF]
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 R. M. David Proposed Mode of Action for In Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract Toxicol Pathol, April 1, 2006; 34(3): 209 - 219. [Abstract] [Full Text] [PDF]
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 R Hauser and A M Calafat PHTHALATES AND HUMAN HEALTH Occup. Environ. Med., November 1, 2005; 62(11): 806 - 818. [Full Text] [PDF]
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 J. C. Corton and P. J. Lapinskas Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract? Toxicol. Sci., January 1, 2005; 83(1): 4 - 17. [Abstract] [Full Text] [PDF]
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 J. J. Schlezinger, G. J. Howard, C. H. Hurst, J. K. Emberley, D. J. Waxman, T. Webster, and D. H. Sherr Environmental and Endogenous Peroxisome Proliferator-Activated Receptor {gamma} Agonists Induce Bone Marrow B Cell Growth Arrest and Apoptosis: Interactions between Mono(2-ethylhexyl)phthalate, 9-cis-Retinoic Acid, and 15-Deoxy-{Delta}12,14-prostaglandin J2 J. Immunol., September 1, 2004; 173(5): 3165 - 3177. [Abstract] [Full Text] [PDF]
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