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Commissariat à l’Energie Atomique, Direction des Sciences du Vivant/Departement de Radiobiologie et Radioprotection/Service d’Etude de la Gamétogenèse et de la Génotoxicité/Laboratory of Differentiation and Radiobiology of the Gonads (R.L., H.C., C.P., R.H., V.R.-F.), Unit of Gametogenesis and Genotoxicity, Institut National de la Santé et de la Recherche Médicale U566 (R.L., H.C., C.P., R.H., V.R.-F.), and University of Paris 7 Denis Diderot (R.L., H.C., C.P., R.H., V.R.-F.), Unité de Formation et Recherche of Biology, F-92265 Fontenay-aux-Roses, France; Assistance Publique-Hôpitaux de Paris (A.-C.D., R.F.), Groupement Hospitalier Universitaire Sud, Hôpital A. Béclère, Service de Gynécologie-Obstétrique, F-92141 Clamart, France; and Institut Federatif de Recherche 13 (R.L., H.C., C.P., A.-C.D., R.F., R.H., V.R.-F.), F-92141 Clamart, France
Address all correspondence and requests for reprints to: Romain Lambrot, Institut National de la Santé et de la Recherche Médicale U 566, Commissariat à l’Energie Atomique, Université Paris 7, Route du Panorama BP 6, 92265 Fontenay-aux-Roses Cedex, France. E-mail: romain.lambrot{at}cea.fr; or virginie.rouiller-fabre{at}cea.fr.
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Abstract |
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Objectives: To identify these factors, we developed an organ culture system for human fetal testes recovered during the first trimester (6–12 wk) of gestation. We first aimed at investigating the characteristics of this system by comparing the in vivo and in vitro gametogenesis and steroidogenesis. Second, we used organ culture to investigate the effect on the human testicular functions of retinoic acid (RA), previously described as a regulator of gonadal development in rodents.
Results: Organ culture proved to be an efficient tool for studying the early development of the testicular functions. Indeed, this system was able to maintain satisfactory development of the germ cells and Leydig cells in the absence of any added factor. For older fetuses, the number of germ cells decreased in culture and the LH was necessary to maintain the steroidogenic activity. The addition of 10–6 M RA decreased the total number of germ cells in the fetal testis at all studied stages. This resulted from an increase in apoptosis, which slightly exceeded the increase of proliferation. However, RA had a stimulatory effect on the steroidogenic function for the youngest fetuses over a short period of time by increasing the expression of P450 cholesterol side-chain cleavage, 17 
-hydroxylase/C17–20 lyase, and steroidogenic acute regulatory protein.
Conclusions: Thus, RA appears as a potential regulator of both gametogenesis and steroidogenesis in human fetal testis. Our organ culture is an interesting tool for studying the effects of various factors on the development of human fetal testis, in particular the effect of hormone-disrupting chemicals.
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Introduction |
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In human, the chronology of the testicular development has also been studied. PGCs start migrating from the stalk of allantois at 4 wk gestation and enter the gonad in the fifth week (7). In males, the PGCs are then enclosed by the differentiating Sertoli cells. Thus, the first morphological sign of testicular differentiation is the formation of testicular cords, which can be seen between 6 and 7 wk gestation (7, 8). The characterization of fetal germ cells was first achieved using electron microscopy and showed the existence of distinct subpopulations (7, 9, 10). Recently an immunohistochemical study using germ cell markers allowed the characterization of three different types of human testicular germ cells: gonocytes, intermediate germ cells, and prespermatogonia (11). However, the changes in the number of germ cells during complete human fetal development and the existence of alternate periods of activity (mitosis and apoptosis) and quiescence have never been reported. The steroidogenic function of human fetal testis is better documented. Steroid-secreting Leydig cells can be seen in the testis at 8 wk gestation (12). Shortly after their appearance, the concentration of androgens in the testicular tissue and blood starts to rise, reaching a maximum at 14–16 wk gestation (13, 14). This increase comes with an increase in the number of Leydig cells (13).
In rodents, there are many factors that regulate these developmental steps. We aimed at developing an organ culture system to study this regulation (15, 16, 17). In this in vitro system, the architecture of the testis and intercellular communications are conserved (18). It also allowed us to mimic the development of Sertoli cells, gonocytes, and Leydig cells that have been observed in vivo with no need to add serum or other factors (15, 16, 17). Using this system, we have shown the importance of vitamin A (retinol) and its biologically active metabolite, retinoic acid (RA), during the development of rat testis (16, 19, 20).
In this present paper, our first aim was to set up an organ culture system to study the development of both gametogenesis and steroidogenesis in human. Then we used this in vitro system to investigate the effects of RA on the development of the human fetal testis because no data are available on the role of RA in the development of the human gonad despite both deficiency and excess of vitamin A being common occurrences (21, 22).
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Materials and Methods |
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Human fetal testes were obtained from pregnant women referred to the Department of Obstetrics and Gynecology at the Antoine Béclère Hospital (Clamart, France), for legally induced abortion in the first trimester of pregnancy [6–12 wk gestation (42–84 d)]. The Antoine Béclère Ethics committee approved this study. The termination of pregnancy was induced by treatment with mifegyne (RU 486, Exelgyn, Paris, France; 200 mg orally) followed by aspiration 48 h later. None of the terminations was for reasons of fetal abnormality, and all fetuses appeared morphologically normal. The fetus was dissected under a binocular microscope, and testes were removed aseptically and were immediately explanted in vitro, or fixed in Bouin’s fluid, or placed in TRIzol reagent (Invitrogen, Cergy-Pontoise, France), depending on the subsequent experiment. 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 (23). We found testes within the abortive material in only 12% of cases.
Organ cultures
Testes were cultured on Millicell-CM Biopore membranes (pore size 0.4 µm; Millipore, Billerica, MA) in Ham F12/DMEM (1:1) (Life Technologies, Inc., Grand Island, NY) containing 80 µg/ml gentamicin (Sigma, St. Louis, MO) as previously described (16). Briefly, testes were cut into small pieces (2, 8, 12, and 16 pieces at 6–7, 8–9, 10, and 11–12 wk gestation, respectively), and all the pieces from the same testis were placed on a single Millicell membrane and floated on 0.3 ml of culture medium in tissue culture dishes and cultured for 4 d at 37 C in a humidified atmosphere containing 95% air-5% CO2. The medium was changed every 24 h. We determined the number of germ cells before and after 4 d of culture by fixing one testis at the time of explantation, whereas the other testis from the same fetus was cultured in medium with no added factor. We measured the response to RA (all-trans, Sigma) by comparing one testis cultured in medium containing RA with the other testis from the same fetus cultured in medium not containing retinoids, as a control. In some cases, for testosterone response to RA, different pieces from the same testis were compared. In some experiments, we added 100 ng/ml LH from human pituitary (
5000 IU/mg) (Sigma) to either the treated medium or both media for the whole culture period or for the last 3 h of culture. For cellular analyzes, the whole explant was fixed for 2 h in Bouin’s fluid and embedded in paraffin, and 5-µm sections were cut. For gene expression, testes were placed in TRIzol reagent.
Germ cell counting
We mounted serial sections on slides, removed the paraffin, and rehydrated the section. We then carried out immunohistochemical assays for anti-Müllerian hormone (AMH). Therefore, the germ cells were identified as AMH-negative cells within the seminiferous cords, whereas the Sertoli cells were identified as AMH-positive cells. Briefly, sections were incubated with 3% H2O2 in distilled water for 10 min to inactivate endogenous peroxidases. The slides were then washed, blocked for 30 min with normal goat serum followed by incubation for 2 h with the anti-AMH polyclonal antibody (1:2000) [generously provided by Dr. N. Di Clemente (Institut National de la Santé et de la Recherche Médicale U782, Clamart, France)]. We detected this primary antibody with a biotinylated goat antirabbit secondary antibody and the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized using 3,3'-diaminobenzidine as a substrate. For all immunohistochemical stainings, negative controls were done by omitting the primary antibody. We counted only one of 10 sections for the 6- to 7-wk-old fetuses and one of 20 sections for later stages but never less than 10 sections equidistantly distributed along the testis. All counts were done using Histolab analysis software (Microvision Instruments, Evry, France). We multiplied the sum of the values obtained for the observed sections of one testis by 10 or 20, respectively, to obtain a crude count per testis. We then used the Abercrombie formula (24) to correct for any double counting due to single cells appearing in two successive sections, and so we obtained a true count. All counts were done blind.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining
We detected apoptotic cells in situ using the TUNEL method, as previously described (16, 17). We mounted six sections equidistantly distributed along the testis on a single slide. Positive controls were treated with DNase I (100 µg/ml; Sigma) for 10 min at room temperature to induce DNA fragmentation in all the nuclei, and negative controls were incubated without terminal deoxynucleotidyl transferase. TUNEL-positive and -nonpositive germ cells were counted in all six sections.
Immunohistochemical staining for cleaved caspase-3
Caspase-3 is involved in most of the apoptotic pathways (25). Therefore, we used it as a second marker of apoptosis. As for the TUNEL staining, we mounted six sections on a single slide and heated the slide in a permeabilization solution [0.05 M Tris (pH 10.6)] for 30 min. The procedure was then the same as for detection of AMH except that the primary antibody was the anticleaved caspase-3 antibody (1:50; Cell Signaling Technology, Beverly, MA). Stained and unstained germ cells were counted in all six sections.
Immunohistochemical staining for Ki67
Ki67 is a nuclear antigen present only in cycling cells (26). Therefore, we used it as a marker of germ cell proliferation. We mounted six sections on a single slide and heated the slide in a permeabilization solution [0.01 M sodium acetate (pH 2)] for 30 min. The procedure was then the same as for detection of AMH except that normal horse serum and biotinylated horse antimouse secondary antibody were used. The monoclonal anti-Ki67 antibody (1:50) was purchased from Dako (Trappes, France).
Double staining using VIP
After the first immunohistochemical staining (TUNEL, cleaved caspase-3, Ki67) revealed with 3,3'-diaminobenzidine, the sections were washed in PBS for 10 min, processed for AMH immunohistochemical staining as previously described, and visualized using VIP as a peroxidase substrate (Vector Laboratories).
Testosterone RIA
We measured the testosterone secreted into the medium in duplicate by RIA as previously described (15). No extraction or chromatography was performed because 17ß-hydroxy-5-androstan-3-one, the only steroid that significantly cross-reacts with testosterone (64%), is secreted in minute amounts by the human fetal testis (27).
Reverse transcription and real-time PCR
We evaluated the expression of P450 cholesterol side-chain cleavage (P450SCC), P450 17-hydroxylase/C17–20 lyase (P450C17), and steroidogenic acute regulatory protein (StAR) in fetal testes by a reverse transcription followed by a real-time PCR (real-time RT-PCR). At the end of the culture period, total RNA was extracted from fetal testes with the TRIzol reagent according to the manufacturer’s instructions. RNAs were quantified by using the RiboGreen RNA quantitation kit (Molecular Probes, Cergy-Pontoise, France), and 50 ng of total testicular RNA was reverse transcribed. The primers and probes used were assays on demand designed by Applied Biosystems (Courtaboeuf, France) (sequences not provided, P450scc, Hs00167984_m1; P450c17, Hs00164375_m1; StAR, Hs00264912_m1; ß-actin, Hs99999903_m1; ARN 18S, Hs99999901_s1). Reactions were carried out and fluorescence was detected on an ABI Prism 7000 apparatus (Applied Biosystems, Foster City, CA). Each sample was run in duplicate, and a control PCR was also carried out with RNA for each sample. Negative controls were run for every primer/probe combination. The measured amount of each cDNA was normalized using ß-actin and ARN 18S to confirm.
Statistical analysis
All values are expressed as means ± SEM. The significance of the differences between mean values for the treated and untreated testes was evaluated using Student’s paired t test. Other means were compared by one-way ANOVA (Tukey-Kramer’s test).
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Results |
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We removed testes from fetuses at about 6 wk of development to 10 wk. For each fetus, we fixed one testis at the time of explantation (Fig. 1
, A and C) and cultured the other testis for 4 d in control medium. After culture (Fig. 1, B and D), the architecture of the testis was well conserved, and Sertoli cells were aggregated in cords and surrounded germ cells. AMH was still expressed at a high level by Sertoli cells. We also observed no sign of necrosis or disorganized areas in the tissue. The germ cells displayed large, spherical nuclei containing two or more globular nucleoli and a clearly visible cytoplasm that corresponds to gonocyte morphology (Fig. 1, A–F).
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We evaluated the number of germ cells and their mitotic and apoptotic indices after 4 d of culture and compared these with the values obtained at the time of explantation. For testes explanted before or at 7 wk, the number of germ cells remained the same before and after culture, whereas it decreased 1.8 times for testis explanted at more than 7 wk (Fig. 2A). The proliferation detected by the immunostaining of Ki67 was increased significantly only for the oldest ones (Fig. 2B). However, we observed cleaved caspase-3-positive germ cells at explantation, and their percentage tended to increase in culture for young fetuses and increased significantly for more than 7-wk fetuses (Fig. 2C).
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We cultured testes from fetuses at 6–12 wk of development for 4 d in control medium, and we measured the daily testosterone secretion directly in the medium. The secretion profiles differed as a function of the age of the fetus at the time of explantation (Fig. 3, A–C). At 6 wk of development, testosterone production increased significantly during the culture period from 12 ± 2.5 pg/testis per hour at d 1 to 36.4 ± 11.5 pg/testis per hour at d 4 (Fig. 3A). For testes explanted from fetuses between 6 and 7 wk of development, testosterone secretion remained constant at about 120 pg/testis per hour for the 4 d of culture (Fig. 3B). For testes from later stages (more than 7 wk to 12 wk), the testosterone production was much higher than for testes explanted at earlier stages of development (9.6 ± 2.3 ng/testis per hour) and greatly and significantly decreased after the first day of culture and more so during the 2 following days (Fig. 3C). We added LH (100 ng/ml) to the culture medium for some of the testes from d 2 until the end of the culture. We then assayed basal or LH-stimulated testosterone production. LH increased the production of testosterone during the whole culture (Fig. 3D), avoiding the Leydig cell dedifferentiation.
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We cultured testes from fetuses at about 6 wk of development to 10 wk with or without 10–6 M of RA for 4 d. RA had no effect on the organization of the testis, and the appearance of all cells was similar to the control. We then evaluated the number of germ cells and their mitotic and apoptotic indices. Irrespective of the age, RA reduced the number of germ cells. Therefore, we pooled the results from different ages (Fig. 4A). RA treatment increased significantly both the mitotic index and percentage of TUNEL-positive and cleaved caspase-3-positive germ cells (Fig. 4, B–D).
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We cultured testes from fetuses under 7 wk of development and older with or without 10–6 M of RA for 4 d. RA treatment increased testosterone production, compared with control testes at d 2 and 3 for testis under 7 wk (Fig. 5A). This stimulating effect disappeared at d 4. For testes from fetuses of more than 7 wk, RA treatment had no effect on steroidogenesis (Fig. 5B). Even when LH (100 ng/ml) was added to the medium of both control and treated testis, RA treatment still had no effect on testosterone production (Fig. 5C).
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We analyzed by real-time RT-PCR the effect of RA on the levels of mRNA for P450SCC, P450C17, and StAR after 2 d of culture (Fig. 6). We used testes from fetuses under 7 wk and after 7 wk because at these stages, testes show RA-stimulated and RA-independent testosterone secretion, respectively. For testes from fetuses under 7 wk, RA treatment increased significantly the mRNA levels of all of these enzymes, whereas for testes from fetuses after 7 wk of development, RA treatment no longer had an effect on the mRNA expression.
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Discussion |
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We previously developed an organ culture system for fetal and neonatal rat testes (15, 16). This system allowed us to demonstrate that several factors, hormones, and vitamins can modulate the proliferation of germ cells and the functional development of somatic cells (16, 17, 20). This system is the most suitable for human due to the limited available quantity of material. Moreover, in human, in vitro studies are the only possible experimental studies. We first aimed at investigating the characteristics of this system by comparing in vivo and in vitro gametogenesis and steroidogenesis. From in vivo studies, it is known that during the first trimester of pregnancy, the number of germ cells, mostly identified as gonocytes (11), increases continuously (28). In our culture system, we observed that germ cell number was maintained for young fetuses (
7 wk). Thus, early differentiating human fetal testis is able to produce the factors needed to sustain gametogenesis. For explanted testes at later stages (older than 7 wk to 12 wk), the number of germ cells was reduced. This is due to increased apoptosis slightly exceeding the increase in the mitotic activity. In our study we determined the total number of germ cells before and after culture, which allows us to characterize the biology of the germ cells in vitro. Previous studies using organ cultures in human determined only the density of germ cells (29) or did not even study this parameter (30). However, the human fetal testis is highly heterogeneous with large variations in germ cell density within the organ (28). Therefore, the number of germ cells needs to be determined for the whole testis.
We also focused on testosterone secretion. We observed three different secretion profiles, depending on the age of the fetuses. For testes from fetuses before 7 wk gestation, testosterone secretion increased during the culture period. This is consistent with in vivo observations. Indeed, testosterone can be detected from 6 to 7 wk gestation (31), and its serum concentration then increases until 14–16 wk gestation (13, 14). Our results also show that early testosterone production does not appear to be controlled by extratesticular factors because nothing was added to the culture medium. This confirms that neither human chorionic gonadotropin (hCG) nor LH control initial Leydig cell differentiation, which is consistent with the study by Kremer et al. (32) on an inactivating LH receptor-mutated patient who displayed some development of the Wolffian ducts. Over a small range of gestational age (
6–7 wk gestation), testosterone secretion remained constant during the culture period in our system. For later developmental stages, testosterone secretion fell. Therefore, an essential factor for testosterone secretion in vitro appeared to be missing. In human, before testicular development, hCG is secreted by the placenta and LH is produced by the pituitary gland as early as the 10th wk (14). Hence, we added LH to the culture medium and observed a constant or even increasing testosterone secretion. So we indeed observed the LH/hCG stimulatory effect that was seen by Huhtaniemi et al. (33) on minces of human fetal testis. These results do not agree with a previous study by Abramovich et al. (34) that showed that hCG did not maintain testosterone secretion in culture. However, our results confirm that hCG/LH is absolutely required for maintaining testosterone secretion after the seventh week (13).
The second purpose of our study was to use this organ culture system to study the effect of RA on human testis development. RA is an important regulator of fetal development in many organs. Previous studies have also shown that retinoids have many effects on the development and/or function of the three main cell types in rodent fetal and neonatal testis (16). In human, there is often a deficiency or excess of vitamin A or retinol. Severe vitamin A deficiency is observed in most developing countries, and an insufficient intake of vitamin A has been reported for 20–25% of adult women in Paris (21). Conversely, hyperretinoidemia is frequently provoked for therapeutic purposes (22). Therefore, we tested the effect of adding 10–6 M of RA on testis in culture. We chose this dose according to our previous studies on rat fetal testis. In addition studies on the endogenous distribution of retinoids during normal development in whole mouse embryos showed that there was a 5- to 10-fold excess in concentration of retinol, compared with RA (35). In human fetuses, there have been no studies on retinol concentration, although Berggren Soderlund et al. (36) showed that human newborns have serum retinol concentration of 10–6 M. Therefore, 10–6 M RA appeared appropriate to produce a clear result without any toxic effect.
Although RA causes disruption of the seminiferous cords in the testis of rat fetuses at 14.5 dpc (16), we observed no disorganization in human testis.
After 3 d of treatment, RA reduced the number of germ cells in human fetal testis. This was also observed in 14.5 dpc rat (16) and 12.5 dpc mouse (Livera, G., and V. Rouiller-Fabre, unpublished data).
We also found that, as in rat, this effect was due to a large increase in apoptosis that greatly exceeded the increase in mitosis. This is consistent with the apoptotic effect of retinoids in many other cell types or lines (37, 38). Whether RA acts on germ cells directly or via autocrine/paracrine factors and the types of receptors and pathways that are implicated remain to be investigated.
The effect of RA on germ cells was reproducible at all stages of development studied, confirming that our in vitro culture system allows us to study the effects of different factors on germ cells from 6 to 12 wk of fetal development.
RA also had a stimulatory effect on the steroidogenic function over a short period of time. This period corresponds to the period in which testosterone secretion spontaneously increased or remained constant in culture without any stimulation. After this period, RA had no effect on testosterone secretion irrespective of whether LH was added to the culture medium. The effect, which was still present after 3 d of culture, disappeared on the fourth day, i.e. the third day of RA treatment. This showed that RA is an early and transient regulator of testosterone secretion.
When RA stimulated testosterone secretion, the levels of three enzymes involved in steroidogenesis were increased, whereas when RA had no effect, the levels of these enzymes remained unchanged. In rat, RA reduces testosterone secretion by only decreasing the level of P450C17 (20).
In conclusion, our study has shown that our in vitro organ culture system is a powerful tool for studying the development of the human fetal testis. It enabled us to show that RA is a potential regulator of germ cell proliferation and steroidogenesis during the first trimester of pregnancy.
Recently several studies have linked male reproductive disorders in human, such as decreasing male fertility (39), increasing incidence of testicular cancer (40), and cryptorchidism and hypospadias (41) with exposure to hormone-disrupting chemicals. These chemicals may alter male fertility by acting early in gonad development. Thus, our organ culture system appears particularly suitable to study the effect of potential toxic substances on human fetal testis development.
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Acknowledgments |
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Footnotes |
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R.L., H.C., C.P., A.-C.D., R.H., and V.R.-F. have nothing to declare, R.F. received lecture fees from different pharmaceutical companies that are not directly related to the material being published.
First Published Online April 18, 2006
Abbreviations: AMH, Anti-Müllerian hormone; dpc, days post conception; hCG, human chorionic gonadotropin; P450C17, P450 17-hydroxylase/C17–20 lyase; PGC, primordial germ cell; P450SCC, P450 cholesterol side-chain cleavage; RA, retinoic acid; StAR, steroidogenic acute regulatory protein; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling.
Received September 23, 2005.
Accepted April 6, 2006.
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-Reduced androgens in the human fetal testis. J Clin Endocrinol Metab 64:628–630[Abstract] and RXR induces differentiation and apoptosis, respectively, in HL-60 cells. Cell Growth Differ 7:179–186[Abstract]This article has been cited by other articles:
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  K. Jahnukainen, J. Ehmcke, M. Nurmio, and S. Schlatt Irradiation Causes Acute and Long-Term Spermatogonial Depletion in Cultured and Xenotransplanted Testicular Tissue from Juvenile Nonhuman Primates Endocrinology, November 1, 2007; 148(11): 5541 - 5548. [Abstract] [Full Text] [PDF]
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 P. A. Fowler, D. R. Abramovich, N. E. Haites, P. Cash, N. P. Groome, A. Al-Qahtani, T. J. Murray, and R. G. Lea Human fetal testis Leydig cell disruption by exposure to the pesticide dieldrin at low concentrations Hum. Reprod., November 1, 2007; 22(11): 2919 - 2927. [Abstract] [Full Text] [PDF]
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 R. Lambrot, H. Coffigny, C. Pairault, C. Lecureuil, R. Frydman, R. Habert, and V. Rouiller-Fabre High Radiosensitivity of Germ Cells in Human Male Fetus J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2632 - 2639. [Abstract] [Full Text] [PDF]
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 J. Merlet, C. Racine, E. Moreau, S. G. Moreno, and R. Habert Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation PNAS, February 27, 2007; 104(9): 3615 - 3620. [Abstract] [Full Text] [PDF]
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