Skip Navigation


Hum. Reprod. Advance Access originally published online on September 21, 2007
Human Reproduction 2007 22(11):2912-2918; doi:10.1093/humrep/dem300
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF ) Freely available
Right arrow All Versions of this Article:
22/11/2912    most recent
dem300v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Coutts, S.M.
Right arrow Articles by Anderson, R.A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coutts, S.M.
Right arrow Articles by Anderson, R.A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Environmental toxicant-induced germ cell apoptosis in the human fetal testis

S.M. Coutts1, N. Fulton1 and R.A. Anderson2,3

1 MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK 2 Division of Reproductive and Developmental Sciences, Centre for Reproductive Biology, The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK

3 Correspondence address. Tel: +44-131-2426386; Fax: +44-131-2426629; E-mail: richard.anderson{at}ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
BACKGROUND: Disorders of the male reproductive system are increasing in prevalence. The term testicular dysgenesis syndrome emphasizes the importance of developmental influences on the aetiology of conditions including cryptorchidism, testicular germ cell cancer and reduced spermatogenesis. Men whose mothers smoked during pregnancy have lower sperm production. Cigarette smoke contains agents acting on the aryl hydrocarbon receptor (AHR). We have investigated the presence of AHR in the developing human testis and the effects of functional activation.

METHODS AND RESULTS: Immunohistochemistry determined AHR to be expressed by germ cells in the human testis between 7 and 19 week gestation, but not by other cells. Treatment of cultured fetal testis with an AHR ligand present in tobacco smoke increased markers of cell apoptosis, and this was prevented by an AHR receptor antagonist. Immunohistochemistry indicated that apoptosis was restricted to germ cells.

CONCLUSIONS: Germ cells in the developing human testis are a target for regulation by AHR ligands. Activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth, and may also be of importance in other conditions comprising the testicular dysgenesis syndrome.

Key words: testis/environmental toxin/spermatogenesis/development/smoking


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Adult male reproductive function is considerably influenced by events in the intrauterine and immediate post-natal periods. The testicular dysgenesis syndrome encompassing abnormalities including cryptorchidism, hypospadias, germ cell cancer and low sperm production in adulthood is believed to arise during this time (Skakkebak et al., 2001Go; Sharpe, 2006Go). The rising prevalence of this syndrome and the marked variation between countries indicate environmental as well as genetic contributions to its aetiology (Boisen et al., 2004Go; Richiardi et al., 2004Go).

Testicular development follows migration of primordial germ cells (PGCs) from the extraembryonic mesoderm of the yolk sac to the indifferent gonad. Sexual differentiation occurs at ~8 weeks gestation (i.e. 6 weeks postconception) in the human, when PGCs become surrounded by Sertoli cells, forming testicular cords, and continue to proliferate throughout fetal life (Hilscher, 1991Go). This increase in number is rapid, with a 10-fold increase in germ cell number in the 3 weeks following sexual differentiation. Sertoli cells proliferate in parallel, maintaining a constant ratio between germ cell and Sertoli cell numbers (Bendsen et al., 2003Go).

This period is therefore critical for the establishment of normal testicular structure and function, and is a period of potential vulnerability of the testes to external influences. One potential external influence is cigarette smoke. It is well established that smoking is associated with a significant reduction in sperm concentration and quality (Vine et al., 1994Go; Kunzle et al., 2003Go). There is also evidence for adverse effects on the developing testis, as epidemiological studies have shown that sons exposed to maternal smoking in utero have a reduction in sperm concentration of between 20 and 48% in comparison to unexposed men (Storgaard et al., 2003Go; Jensen et al., 2004Go). Tobacco smoke contains polycyclic aromatic hydrocarbons (PAHs), environmental toxicants produced primarily as a by-product of fossil fuel combustion. PAHs bind to and activate the aryl hydrocarbon receptor (AHR) (Hankinson, 1995Go), which is a member of the per-Arnt-Sim family of transcriptional factors. The AHR is involved in toxin metabolism and also has physiological roles in the development of several organ systems (Pocar et al., 2005Go). An endogenous ligand has not been identified.

Treatment of fetal mice in utero with the AHR ligand benzo(a)pyrene reduced the fertility of both male and females in adulthood (Mackenzie and Angevine, 1981Go). Maternal exposure to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), also an AHR ligand, has also been demonstrated to have irreversible effects on the developing reproductive tract in rats including reduced sperm production (Mably et al., 1992aGo; Gray et al., 1995Go, 1997Go; Sommer et al., 1996Go; Wilker et al., 1996Go; Faqi et al., 1998Go), and reduced the size of the reproductive organs (Mably et al., 1992bGo; Bjerke and Peterson, 1994Go; Roman et al., 1995Go, 1998Go; Gray et al., 1997Go; Roman and Peterson, 1998Go). Both in vitro and in vivo exposure of mouse fetal ovaries to PAHs results in an increase in germ cell apoptosis, subsequently demonstrated to be mediated through an increase in Bax expression (Matikainen et al., 2001Go, 2002Go).

The aim of this study was therefore to explore the expression, distribution and effect of functional activation of the AHR in the human fetal testis to investigate mechanisms underlying the effects of environmental AHR agents on testicular development. This study investigated expression of Bax, cleaved caspase 3 and cleaved PARP, which are established markers of the regulation, execution and a target of the apoptotic pathway, respectively, (Gross et al., 1999Go); n the case of Bax, a link with AHR activation in the developing gonad has been previously demonstrated (Matikainen et al., 2001Go).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Tissue
Human fetal testes were obtained following medical termination of pregnancy during both the first and second trimesters (64 days to 20 week gestational age). Women gave consent according to national guidelines and the study was approved by the Lothian Research Ethics Subcommittee (Polkinghorne, 1989Go). Termination of pregnancy was induced by treatment with mifepristone (200 mg orally) followed 48 h later by misoprostal (800 mcg) three hourly per vaginum. None of the terminations were for reasons of fetal abnormality, and all fetuses appeared morphologically normal. Gestational age was determined by ultrasound examination before termination and confirmed by subsequent direct measurement of foot length.

Testes were dissected and either snap frozen and stored at –70°C, fixed in Bouins for 2 h, followed by processing for immunohistochemistry or further dissected for in vitro culture experiments.

Immunohistochemistry
Paraffin-embedded testes were cut into 5-µm sections and mounted onto electrostatically charged microscope slides (VWR, Poole, UK), dried overnight and then dewaxed and rehydrated using conventional methods. Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 min at room temperature. After a wash in water, slides were transferred into Tris-buffered saline [TBS; 0.05 M Tris, 0.85% NaCl (pH 7.6)] for 5 min and blocked for 30 min in normal serum (Diagnostics Scotland, Carluke, UK) diluted 1:4 in TBS containing 5% BSA. Sections were blocked with avidin (0.01 M; 15 min) and then with biotin (0.001 M; 15 min; both from Vector Laboratories, Peterborough, UK) with washes in TBS in between. Primary antibodies used were rabbit polyclonal immunoglobulin G IgG anti-Bax (Santa Cruz, CA, USA) and rabbit polyclonal anti-cleaved caspase 3 (New England Biolabs, Herts., UK). Antibodies were diluted 1:100 and 1:50, respectively. For detection of cleaved caspase 3, antigen retrieval was effected by boiling in citrate (0.01 M) for 2 min followed by immediate cooling in cold water. Sections were incubated in the respective antibodies overnight at 4°C. Bound antibody was detected using a goat anti-rabbit biotinylated secondary antibody diluted at 1:500 (Dako, Glostrup, Denmark) for 30 min at room temperature followed by incubation in avidin/biotin horse-radish peroxidese (HRP)-linked complex (Dako) for 30 min at room temperature according to the manufacturer’s instructions. Visualization of the bound antibody was realized using 3,3'-diaminobenzidine tetrahydrochloride (Dako). Sections were counterstained using hematoxylin, dehydrated, mounted and visualized by light microscopy. Images were captured using an Olympus Provis microscope (Olympus Optical Co., London, UK) equipped with Kodak DCS330 camera (Eastman Kodak Co., Rochester, NY, USA). Negative controls were incubated with rabbit IgG in place of primary antisera.

Immunofluorescence
Paraffin-embedded testes were cut into 5-µm sections and mounted onto electrostatically charged microscope slides (VWR), dried overnight and then dewaxed and rehydrated using conventional methods. Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 min at room temperature. After a wash in water, slides were transferred into phospho-buffered saline (PBS) (Sigma, Dorset, UK) for 5 min and blocked for 30 min in normal serum (Diagnostics Scotland) diluted 1:4 in PBS containing 5% BSA. Sections were blocked with avidin (0.01 M; 15 min) and then biotin (0.001 M; 15 min; both from Vector Laboratories) with washes in PBS in between. AHR antibody (Affinity BioReagents, Cambridge, UK) was diluted 1:150 and applied to sections at 4°C overnight in a humidified chamber. AHR was visualized by tyramide-enhanced fluorescein via a peroxidase conjugated goat anti-mouse secondary antibody diluted 1:200. Sections were counterstained with propidium iodide 1:1000. Fluorescent images were captured using a LSM510 confocal microscope. Negative controls were incubated with mouse IgG, omitting primary antisera.

Tissue culture
Fetal testes were dissected and cultured in minimum essential medium {alpha} with phenol red (GibcoBRL, Life Technologies, Paisley, UK) supplemented with 2 mM pyruvate, 2 mM glutamine, 1x ITS supplement (Sigma) and 3 mg/ml BSA and with penicillin, streptomycin and amphotericin as previously described (Robinson et al., 2003Go). Fetal testes (14–19 weeks) were dissected in prewarmed and gassed medium to yield tissue fragments ~1 mm3. Between six and eight fragments were transferred in 500 µl medium to wells containing inserts (Millicell-CM; Millipore UK Ltd, Watford, UK) in a 24-well tissue culture plate such that tissue fragments were supported at the meniscus of the medium. In the initial experiments, tissues were treated with appropriately diluted acetone solvent (controls) or the AHR ligand 9,10-dimethyl-1,2-benzanthracene-3,4-dihydrodiol (DMBA-DHD, NCI Chemical Carcinogen Reference Standards Repository, USA), 0.1 or 1 µM, for 24 h.

Further culture experiments were performed as above with tissues treated for 24 h with controls, 1 µM DMBA-DHD, 1 µM alpha-naphthoflavone ({alpha}NF, Sigma) which is a selective antagonist for AHR, or 1 µM DMBA-DHD + 1 µM {alpha}NF.

Fragments of testes were collected post-culture, washed in PBS and then either snap frozen and stored at –70°C, for immunoblotting, or fixed in Bouins for 2 h, followed by processing for immunohistochemistry.

Immunoblotting
Fetal testes were homogenized in lysis buffer containing 80 mM Tris (pH6.8), 1% (v/v) glycerol, 1% (w/v) sodium dodecyl sulphate (SDS) and a Complete Mini protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). Total protein quantification was performed using the BioRad protein assay kit.

Proteins were boiled in a 1:3 volume of 4x reduced sample buffer [20% 250 mM Tris (pH6.8), 4% SDS, 10% β-mercaptoethanol and 0.1% bromophenol blue]. Samples of 10 µg total protein was loaded onto 15% SDS–polyacrylamide gel electrophoresis (PAGE) gels with high-molecular weight Rainbow protein markers run in parallel (Amersham Biosciences, Buckinghamshire, UK). Gels were blotted onto polyvinyl difluoride membrane (Amersham Biosciences) and then blocked in TBS and Tween [10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20] containing 5% powdered milk before overnight incubation at 4°C with primary antibody. Rabbit polyclonal antibodies to Bax (Santa Cruz), cleaved PARP (Cell Signaling Technology, Danvers, USA) and β-actin (Santa Cruz) were diluted 1:1000, 1:1000 and 1:100 in TBS and Tween 20 containing 5% powdered milk. Bound antibody was detected using HRP-linked secondary antibodies diluted 1:10 000 (Cell Signaling Technology), and proteins were revealed and quantified by phosphoimager analysis using the Typhoon 9400 system (Amersham Biosciences) and normalized for protein loading using β-actin.

Statistics
Results are expressed as mean ± SEM. The effects of DMBA-DHD and {alpha}NF on Bax and cleaved PARP expression relative to β-actin were analysed by ANOVA with Tukey–Kramer post hoc testing.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
AHR protein in human fetal testis
Expression of AHR was detected by immunofluorescence in all specimens of human fetal testis examined, between the gestational ages of 64 days and 20 weeks (Fig. 1A and B). AHR was exclusively localized to the cytoplasm of germ cells, with no expression by other cell types in either the testis cords or interstitium at any gestation examined. In the first trimester, AHR was expressed by all germ cells, however, by the second trimester, AHR was only expressed by a subpopulation of germ cells. Immunoblotting confirmed the expression of AHR protein in the human fetal testis, with a single band detected of molecular weight 95 kDa (Fig. 1C).


Figure 1
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1: Expression of AHR in fetal testis

Immunofluorescent localization of AHR in human fetal testis at (A) 64 days and (B) 18 weeks gestation. Insert represents negative control. White arrows indicate AHR expression in the cytoplasm of germ cells. Arrowhead indicates germ cells not expressing AHR. (C) Detection of AHR by Western immunoblot in human fetal testis, with a single band of 95kDa detected. Weeks of gestation are indicated

 
Bax and cleaved caspase 3 expression in fetal testis
Apoptotic cells in the testis were identified by immunolocalization of Bax and cleaved caspase 3. Bax was expressed in all specimens of human fetal testis examined, being located in both the Sertoli and germ cells, with no expression in any cells of the interstitium (Fig. 2C). An apparent increase in Bax expression was observed in germ cells in human fetal testis cultured with DMBA-DHD (Fig. 2G).


Figure 2
View larger version (89K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2: Effect of AHR activation on Bax and cleaved caspase 3 expression in fetal testis

H&E stain of uncultured 16 weeks human fetal testis (A); H&E stain of cultured 16 week human fetal testis (B). Bax localization in uncultured 16 weeks human fetal testis (C); 16 weeks testis control culture (E); +1 µM DMBA-DHD (G); +1 µM {alpha}NF (I) and +1 µM DMBA-DHD plus 1 µM {alpha}NF (K). The arrowhead indicates Bax expression by germ cells; the arrows indicate germ cells not expressing Bax. Cleaved caspase 3 localization in uncultured 16 weeks human fetal testis (D); 16 weeks testis control culture (F); +1 µM DMBA-DHD (H); +1 µM {alpha}NF (J) and +1 µM DMBA-DHD plus 1 µM {alpha}NF (L). The arrowhead demonstrates cleaved caspase 3 expression by germ cells; the arrows indicate germ cells not expressing cleaved caspase 3. Scale bars 50 µm in B, 30 µm in A, D and F and 20 µm in C, E and G–L

 
Cleaved caspase 3 is another marker of apoptosis. Immunohistochemical examination of uncultured human fetal testes and human fetal testis cultured with the vehicle, revealed no cleaved caspase 3 expression (Fig. 2D and F). Testis cultured with DMBA-DHD however showed expression of cleaved caspase 3 in a small number of cells in the testicular cords (Fig. 2H). Morphologically, these cells appeared to be germs cells, with no other cell types in either the testis cords or interstitium expressing cleaved caspase 3.

PAHs increase apoptosis in fetal testis
The effect of PAHs on markers of apoptosis in the fetal testis was investigated using an in vitro culture system and immunoblot analysis. Preliminary experiments demonstrated that DMBA-DHD treatment increased Bax expression by ~2-fold in a concentration dependent manner (P = 0.01, Fig. 3A). Further experiments again resulted in a 2-fold increase in Bax expression from 6.9 ± 1.9 to 14 ± 1.7 relative to β-actin (P < 0.001, Fig. 3B). Treatment of fetal testis with 1 µM {alpha}NF had no effect alone but completely reversed this proapoptotic effect of DMBA-DHD in germ cells (Fig. 3B and C; 8.3 ± 3.0 {alpha}NF alone, 8.3 ± 1.0 {alpha}NF with DMBA-DHD, P = 0.02 versus DMBA-DHD alone, ns versus control, n = 5).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3: AHR activation increases Bax expression in human fetal testis

(A) Concentration dependent increase in Bax expression after treatment with DMBA-DHD (24 h; 14–19 week gestation, n = 5). (B) Fetal testis was treated with 1 µM DMBA-DHD alone or in combination with 1 µM {alpha}NF, or {alpha}NF alone (24 h; 14–19 week gestation, n = 5). Different superscripts indicate statistical significance: a versus b (P ≤ 0.02); mean ± SEM, n = 5. (C) Western immunoblot of Bax expression in cultured fetal testis. D, DMBA-DHD. Approximate molecular weights (kDa) of the proteins detected are indicated

 
Expression of a further marker of apoptosis, the caspase product cleaved PARP, showed a 3-fold increase from 1.08 ± 0.38 to 3.12 ± 0.5 relative to β-actin (P = 0.02) (Fig. 4). Treatment of fetal testis with 1 µM {alpha}NF had no effect alone but completely reversed the proapoptotic effect of DMBA-DHD in germ cells (Fig. 4) (1.8 ± 0.5 {alpha}NF alone, 1.54 ± 0.5 {alpha}NF with DMBA-DHD, P = 0.03 versus DMBA-DHD alone, ns versus control, n = 5).


Figure 4
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4: AHR activation increases cleaved PARP expression in human fetal testis

Fetal testis was treated with 1 µM DMBA-DHD alone or in combination with 1 µM {alpha}NF (24 h; 14–19 week gestation, n = 5). Different superscripts indicate statistical significance: a versus b. P ≤ 0.03, mean ± SEM

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
This study provides direct evidence that exposure to toxins found in tobacco smoke results in germ cell apoptosis in the developing human testis. Epidemiological studies have demonstrated that sperm production is dose-dependently reduced by 20–48% in the sons of mothers who smoked during pregnancy, and testicular volume is also reduced (Storgaard et al., 2003Go; Jensen et al., 2004Go). Fecundability was also reduced in men exposed to cigarette smoke in utero in a study of 430 Danish couples (Jensen et al., 1998Go), although a previous study found no such effect (Baird and Wilcox, 1986Go).

The results from these experiments demonstrate expression of AHR in germ cells but not in other cell types in first and second trimester human fetal testis. AHR has been previously demonstrated in adult human testis (Schultz et al., 2003Go), with widespread expression in almost all interstitial and tubular cells. Expression fell with increasing gestation, with the AHR expressed in all germ cells in the first trimester but only in a subpopulation in the second trimester. This may indicate that the AHR is only expressed at certain developmental stages as PGCs mature to gonocytes/prespermatogonia (Gaskell et al., 2004Go). The AHR is an orphan receptor member of the per-Arnt-Sim family of transcriptional factors. It is involved in toxin metabolism, but there is also a substantial body of evidence regarding its role in embryological development of different organ systems, which may reflect a physiological function (Pocar et al., 2005Go). AHR has also been implicated as playing a role in normal vascular biology and AHR activation modulates transcription of genes involved in regulating the cell cycle, apoptosis and oxidative stress (Denison and Heath-Pagliuso, 1998Go). In males, knockout of the AHR resulted in reduced weight of the testes and other organs, although adult sperm production was normal (Lin et al., 2003Go). In female mice, knockout of the AHR resulted in an increase in primordial follicles between Days 2–4, suggesting a potential physiological role of the AHR in prenatal germ cell apoptosis (Benedict et al., 2000Go). The data shown here demonstrate highly selective expression of AHR in only the germ cells of the developing human testis during both the first and second trimesters, giving a stage of development during which any physiological role of AHR may be important. However, it also provides a basis for potentially adverse effects by environmental toxicants that act as AHR ligands. These include dioxin and PAHs produced in cigarette smoke.

Human fetal testis was cultured in the presence of DMBA-DHD, the active metabolite of the PAH. DMBA that is produced primarily in cigarette smoke was used to investigate whether AHR activation resulted in apoptosis. Three markers of apoptosis were utilized: Bax, a proapoptotic member of the Bcl-2 family of cell death regulators, cleaved caspase 3 and the caspase target PARP. DMBA-DHD resulted in an increase in expression of Bax and cleaved PARP, which was prevented by the use of the AHR antagonist {alpha}NF (Merchant et al., 1990Go), demonstrating mediation of this effect via the AHR receptor. We were unable to demonstrate expression of cleaved caspase 3 by immunohistochemistry in uncultured tissue or by immunoblotting, which may reflect very low expression, but clear staining in a small number of cells was detected after culture with DMBA-DHD. Morphologically, the cells affected by apoptosis appeared to be germ cells. These data therefore indicate that PAHs acting through the AHR induce apoptosis of germ cells in the developing human testis.

A PAH induced increase in germ cell apoptosis via an increase in Bax expression has been demonstrated in the mouse fetal ovary in vitro, and in utero exposure of female mice also resulted in marked loss of germ cells (Matikainen et al., 2001Go, 2002Go). This has not been previously demonstrated in the male, either rodent or human, although there is considerable evidence as to the adverse effects of in utero or lactational exposure to AHR ligands. In rats, maternal exposure to TCDD causes reduced sperm production (Mably et al., 1992aGo; Gray et al., 1995Go, 1997Go; Sommer et al., 1996Go; Wilker et al., 1996Go; Faqi et al., 1998Go), and reduced volume of the testes and accessory organs (Mably et al., 1992bGo; Bjerke and Peterson, 1994Go; Roman et al., 1995Go, 1998Go; Gray et al., 1997Go). Treatment of adult rodent testis with the AHR ligands benzo(a)pyrene and TCDD resulted in increased apoptosis in seminiferous tubules (Merchant et al., 1990Go; Denison and Heath-Pagliuso, 1998Go; Benedict et al., 2000Go; Revel et al., 2001Go; Lin et al., 2003Go; Schultz et al., 2003Go; Gaskell et al., 2004Go).

Germ cell tumours are believed to arise from fetal germ cells that have not progressed through the normal stages of development, and continue to express markers such as OCT4 (Jones et al., 2004Go). We have previously demonstrated OCT4 expression by germ cells at similar gestations to those expressing AHR (Gaskell et al., 2004Go). The prevalence of testicular germ cell tumours is rising in most developed countries. This has been proposed to be part of the testicular dysgenesis syndrome, also comprising testicular maldescent, hypospadias and disorders of spermatogenesis manifesting as infertility in adult life, all of which are thought to be manifestations of perturbed prenatal development and associated with exposure to environmental toxicants (Skakkebak et al., 2001Go; Sharpe, 2006Go). The environmental ‘hormone disruptor’ 4-octylphenol (4-OPL) has been demonstrated to markedly reduce germ cell number in the fetal human testis in vitro (Bendsen et al., 2001Go). 4-OPL has estrogenic activity and estrogen receptors are expressed by germ cells in the human fetal testis (Gaskell et al., 2003Go). Interactions between AHR and estrogen receptor activation and degradation are increasingly recognized (Ohtake et al., 2003Go; Hockings et al., 2006Go; Ohtake et al., 2007Go) and may provide a common pathway between these data.

In conclusion, the present data demonstrate that germ cells in the developing human testis are a target for regulation by AHR ligands. In addition to unknown physiological processes, activation of AHR by environmental toxicants and AHR-induced apoptotic pathways may be the mechanism of action underlying the epidemiological findings of reduced spermatogenesis in men exposed to cigarette smoke before birth.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
This work was funded by the Medical Research Council.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
We are grateful to Joan Creiger and the staff of the Bruntsfield Suite, Royal Infirmary of Edinburgh, for their considerable assistance in patient recruitment.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Baird DD, Wilcox AJ. Future fertility after prenatal exposure to cigarette smoke. Fertil Steril (1986) 46:368–372.[Web of Science][Medline]

Bendsen E, Laursen S, Olesen C, Westergaard L, Andersen C, Byskov A. Effect of 4-octylphenol on germ cell number in cultured human fetal gonads. Hum Reprod (2001) 16:236–243.[Abstract/Free Full Text]

Bendsen E, Byskov AG, Laursen SB, Larsen HP, Andersen CY, Westergaard LG. Number of germ cells and somatic cells in human fetal testes during the first weeks after sex differentiation. Hum Reprod (2003) 18:13–18.[Abstract/Free Full Text]

Benedict JC, Lin T-M, Loeffler IK, Peterson RE, Flaws JA. Physiological role of the aryl hydrocarbon receptor in mouse ovary development. Toxicol Sci (2000) 56:382–388.[Abstract/Free Full Text]

Bjerke DL, Peterson RE. Reproductive toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male rats: different effects of in utero versus lactational exposure. Toxicol Appl Pharmacol (1994) 127:241–249.[CrossRef][Web of Science][Medline]

Boisen KA, Kaleva M, Main KM, Virtanen HE, Haavisto AM, Schmidt IM, Chellakooty M, Damgaard IN, Mau C, Reunanen M, et al. Difference in prevalence of congenital cryptorchidism in infants between two nordic countries. Lancet (2004) 363:1264–1269.[CrossRef][Web of Science][Medline]

Denison MS, Heath-Pagliuso S. The ah receptor: a regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull Environ Contam Toxicol (1998) 61:557–568.[CrossRef][Web of Science][Medline]

Faqi AS, Dalsenter PR, Merker HJ, Chahoud I. Reproductive toxicity and tissue concentrations of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation. Toxicol Appl Pharmacol (1998) 150:383–392.[CrossRef][Web of Science][Medline]

Gaskell TL, Robinson LL, Groome NP, Anderson RA, Saunders PT. Differential expression of two estrogen receptor beta isoforms in the human fetal testis during the second trimester of pregnancy. J Clin Endocrinol Metab (2003) 88:424–432.[Abstract/Free Full Text]

Gaskell TL, Esnal A, Robinson LL, Anderson RA, Saunders PT. Immunohistochemical profiling of germ cells within the human fetal testis: identification of three subpopulations. Biol Reprod (2004) 71:2012–2021.[Abstract/Free Full Text]

Gray LE, Kelce WR, Monosson E, Ostby JS, Birnbaum LS. Exposure to tcdd during development permanently alters reproductive function in male long-evans rats and hamsters: reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol Appl Pharmacol (1995) 131:108–118.[CrossRef][Web of Science][Medline]

Gray JLE, Ostby JS, Kelce WR. A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male long evans hooded rat offspring. Toxicol Appl Pharmacol (1997) 146:11–20.[CrossRef][Web of Science][Medline]

Gross A, Mcdonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev (1999) 13:1899–1911.[Free Full Text]

Hankinson O. The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol (1995) 35:307–340.[CrossRef][Web of Science][Medline]

Hilscher W. The genetic control and germ cell kinetics of the female and male germ line in mammals including man. Hum Reprod (1991) 6:1416–1425.[Abstract/Free Full Text]

Hockings JK, Thorne PA, Kemp MQ, Morgan SS, Selmin O, Romagnolo DF. The ligand status of the aromatic hydrocarbon receptor modulates transcriptional activation of brca-1 promoter by estrogen. Cancer Res (2006) 66:2224–2232.[Abstract/Free Full Text]

Jensen TK, Henriksen TB, Hjollund NH, Scheike T, Kolstad H, Giwercman A, Ernst E, Bonde JP, Skakkebaek NE, Olsen J. Adult and prenatal exposures to tobacco smoke as risk indicators of fertility among 430 danish couples. Am J Epidemiol (1998) 148:992–997.[Abstract/Free Full Text]

Jensen TK, Jorgensen N, Punab M, Haugen TB, Suominen J, Zilaitiene B, Horte A, Andersen A-G, Carlsen E, Magnus O, et al. Association of in utero exposure to maternal smoking with reduced semen quality and testis size in adulthood: a cross-sectional study of 1,770 young men from the general population in five european countries. Am J Epidemiol (2004) 159:49–58.[Abstract/Free Full Text]

Jones TD, Ulbright TM, Eble JN, Cheng L. Oct4: a sensitive and specific biomarker for intratubular germ cell neoplasia of the testis. Clin Cancer Res (2004) 10:8544–8547.[Abstract/Free Full Text]

Kunzle R, Mueller MD, Hanggi W, Birkhauser MH, Drescher H, Bersinger NA. Semen quality of male smokers and nonsmokers in infertile couples. Fertil Steril (2003) 79:287–291.[CrossRef][Web of Science][Medline]

Lin T-M, Rasmussen NT, Moore RW, Albrecht RM, Peterson RE. Region-specific inhibition of prostatic epithelial bud formation in the urogenital sinus of c57bl/6 mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci (2003) 76:171–181.[Abstract/Free Full Text]

Mably TA, Bjerke DL, Moore RW, Gendron-Fitzpatrick A, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability. Toxicol Appl Pharmacol (1992a) 114:118–126.[CrossRef][Web of Science][Medline]

Mably TA, Moore RW, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol Appl Pharmacol (1992b) 114:97–107.[CrossRef][Web of Science][Medline]

Mackenzie KM, Angevine DM. Infertility in mice exposed in utero to benzo(a)pyrene. Biol Reprod (1981) 24:183–191.[Abstract]

Matikainen T, Perez GI, Jurisicova A, Pru JK, Schlezinger JJ, Ryu HY, Laine J, Sakai T, Korsmeyer SJ, Casper RF, et al. Aromatic hydrocarbon receptor-driven bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Nat Genet (2001) 28:355–360.[CrossRef][Web of Science][Medline]

Matikainen TM, Moriyama T, Morita Y, Perez GI, Korsmeyer SJ, Sherr DH, Tilly JL. Ligand activation of the aromatic hydrocarbon receptor transcription factor drives bax-dependent apoptosis in developing fetal ovarian germ cells. Endocrinology (2002) 143:615–620.[Abstract/Free Full Text]

Merchant M, Arellano L, Safe S. The mechanism of action of [alpha]-naphthoflavone as an inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cyp1a1 gene expression. Arch Biochem Biophys (1990) 281:84–89.[CrossRef][Web of Science][Medline]

Ohtake F, Takeyama K-I, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K, Tohyama C, Krust A, Mimura J, Chambon P, et al. Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature (2003) 423:545–550.[CrossRef][Medline]

Ohtake F, Baba A, Takada I, Okada M, Iwasaki K, Miki H, Takahashi S, Kouzmenko A, Nohara K, Chiba T, et al. Dioxin receptor is a ligand-dependent e3 ubiquitin ligase. Nature (2007) 446:562–566.[CrossRef][Medline]

Pocar P, Fischer B, Klonisch T, Hombach-Klonisch S. Molecular interactions of the aryl hydrocarbon receptor and its biological and toxicological relevance for reproduction. Reproduction (2005) 129:379–389.[Abstract/Free Full Text]

Polkinghorne J. Review of the Guidance on the Research use of Fetuses and Fetal Material. (1989) London: HMSO.

Revel A, Raanani H, Younglai E, Xu J, Han R, Savouret JF, Casper RF. Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo(a)pyrene. Reprod Toxicol (2001) 15:479–486.[CrossRef][Web of Science][Medline]

Richiardi L, Bellocco R, Adami HO, Torrang A, Barlow L, Hakulinen T, Rahu M, Stengrevics A, Storm H, Tretli S, et al. Testicular cancer incidence in eight northern european countries: secular and recent trends. Cancer Epidemiol Biomarkers Prev (2004) 13:2157–2166.[Abstract/Free Full Text]

Robinson LLL, Townsend J, Anderson RA. The human fetal testis is a site of expression of neurotrophins and their receptors: regulation of the germ cell and peritubular cell population. J Clin Endocrinol Metab (2003) 88:3943–3951.[Abstract/Free Full Text]

Roman BL, Peterson RE. In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. Toxicol Appl Pharmacol (1998) 150:240–253.[CrossRef][Web of Science][Medline]

Roman BL, Sommer RJ, Shinomiya K, Peterson RE. In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: impaired prostate growth and development without inhibited androgen production. Toxicol Appl Pharmacol (1995) 134:241–250.[CrossRef][Web of Science][Medline]

Roman BL, Timms BG, Prins GS, Peterson RE. In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development: 2. Effects on growth and cytodifferentiation. Toxicol Appl Pharmacol (1998) 150:254–270.[CrossRef][Web of Science][Medline]

Schultz R, Suominen J, Varre T, Hakovirta H, Parvinen M, Toppari J, Pelto-Huikko M. Expression of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator messenger ribonucleic acids and proteins in rat and human testis. Endocrinology (2003) 144:767–776.[Abstract/Free Full Text]

Sharpe RM. Pathways of endocrine disruption during male sexual differentiation and masculinization. Best Pract Res Clin Endocrinol Metab (2006) 20:91–110.[CrossRef][Medline]

Skakkebak NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects: opinion. Hum Reprod (2001) 16:972–978.[Abstract/Free Full Text]

Sommer RJ, Ippolito DL, Peterson RE. In uteroand lactational exposure of the male holtzman rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: Decreased epididymal and ejaculated sperm numbers without alterations in sperm transit rate. Toxicol Appl Pharmacol (1996) 140:146–153.[CrossRef][Web of Science][Medline]

Storgaard L, Bonde JP, Ernst E, Spano M, Andersen CY, Frydenberg M, Olsen J. Does smoking during pregnancy affect sons’ sperm counts? Epidemiology (2003) 14:278–286.[CrossRef][Web of Science][Medline]

Vine MF, Margolin BH, Morrison HI, Hulka BS. Cigarette smoking and sperm density: a meta-analysis. Fertil Steril (1994) 61:35–43.[Web of Science][Medline]

Wilker C, Johnson L, Safe S. Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring. Toxicol Appl Pharmacol (1996) 141:68–75.[Web of Science][Medline]

Submitted on March 30, 2007; resubmitted on June 29, 2007; accepted on July 17, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Clin. Endocrinol. Metab.Home page
P. A. Fowler, S. Cassie, S. M. Rhind, M. J. Brewer, J. M. Collinson, R. G. Lea, P. J. Baker, S. Bhattacharya, and P. J. O'Shaughnessy
Maternal Smoking during Pregnancy Specifically Reduces Human Fetal Desert Hedgehog Gene Expression during Testis Development
J. Clin. Endocrinol. Metab., February 1, 2008; 93(2): 619 - 626.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF ) Freely available
Right arrow All Versions of this Article:
22/11/2912    most recent
dem300v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (3)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Coutts, S.M.
Right arrow Articles by Anderson, R.A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Coutts, S.M.
Right arrow Articles by Anderson, R.A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?