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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1860
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 2 619-626
Copyright © 2008 by The Endocrine Society

Maternal Smoking during Pregnancy Specifically Reduces Human Fetal Desert Hedgehog Gene Expression during Testis Development

Paul A. Fowler, Sarah Cassie, Stewart M. Rhind, Mark J. Brewer, J. Martin Collinson, Richard G. Lea, Paul J. Baker, Siladitya Bhattacharya and Peter J. O’Shaughnessy

Departments of Obstetrics and Gynaecology (P.A.F., S.C., S.B.) and Biomedical Sciences (J.M.C.), Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom; Biomathematics and Statistics Scotland (M.J.B.), Animal Ecology (S.M.R.), Macaulay Institute, Aberdeen AB15 8QH, United Kingdom; School of Veterinary Medicine and Sciences (R.G.L.), University of Nottingham, Loughborough LE12 5RD, United Kingdom; and Department of Veterinary Preclinical Studies (P.J.B., P.J.O.), Division of Veterinary Physiology and Pharmacology, University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom

Address all correspondence and requests for reprints to: Dr. Paul Fowler, Ph.D., Institute of Medical Sciences, OBGYN, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom. E-mail: p.a.fowler{at}abdn.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Maternal cigarette smoking during gestation increases cryptorchidism and hypospadias and reduces testis size and fertility in sons by unknown mechanisms.

Objective: The objective of the study was to determine whether maternal smoking is linked with changes in male human fetal endocrinology, testis gene expression, and liver concentrations of cigarette smoke chemicals.

Design: This was an observational study of the male fetus, comparing pregnancies during which the mothers either did or did not smoke.

Setting: The study was conducted at the universities of Aberdeen, Glasgow, and Nottingham and Macaulay Institute (Aberdeen).

Patients/Participants: Testes, blood, and livers were collected from 69 morphologically normal human male fetuses of women undergoing elective termination of normal second-trimester pregnancies.

Main Outcome Measures: Testosterone, human chorionic gonadotropin, LH, and cotinine; expression of 30 reproductive/developmental genes; liver concentrations of 16 polycyclic aromatic hydrocarbons; and Leydig, Sertoli. and germ cell numbers were determined.

Results: There were no significant differences in fetal size, testis weight, cell numbers, seminiferous tubule diameter, or circulating LH and testosterone. Fetuses from smoking mothers had smoking range cotinine levels and liver concentrations of polycyclic aromatic hydrocarbons that were significant predictors of maternal smoking (P < 0.001). Only the Sertoli cell-specific gene, desert hedgehog (DHH), was significantly altered by maternal smoking (reduced 1.8-fold, P = 0.013).

Conclusions: The consequences of reduced DHH signaling in men and mice are consistent with epidemiology for effects of gestational maternal smoking on sons. Given the absence of other observed effects of maternal smoking, we concluded that reduced DHH is part of a mechanism linking maternal gestational smoking with impaired reproductive development in male offspring.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
Maternal cigarette smoking during gestation has been increasingly associated with rising incidences of cryptorchidism (1, 2, 3, 4) and hypospadias (5) and reductions in testis size (6), sperm counts/quality (7, 8, 9), and fertility (2, 10, 11) in male offspring. Although not all studies have replicated all these findings (12, 13), the rising incidences of these reproductive problems are associated with testis dysgenesis syndrome (14, 15, 16), which has its origin during fetal development. The only component of testis dysgenesis syndrome that does not seem to be associated with gestational maternal smoking (17, 18) is testicular cancer (also increasing in incidence), although a strong geographical association exists between the two (19). Maternal smoking during gestation disturbs reproductively important developmental events, including testosterone production (20), the fetal hypothalamic-pituitary-adrenal axis (21), and neonatal prolactin and GH (22) levels. It should be noted that some of these data (e.g. Ref. 20) have not yet been replicated and, interestingly, adult sons of gestational smokers do not appear to have significantly disturbed endocrine profiles (23). However, gestational maternal smoking is also associated with many developmental problems, including impaired fetal growth (24, 25), which is largely assumed to be due to poor placental development; childhood respiratory ill health (26); and abnormalities of the nervous system and cognition (27) as well as possible increased mortality risk for male fetuses (28).

Cigarette smoke contains a complex mix of chemicals that can affect fetal development, including metals (29), nicotine (30), and polycyclic aromatic hydrocarbons (PAHs) (31). The latter are a large class of toxic pollutants that are mutagenic (32) and cross the placenta, thereby increasing fetal susceptibility to DNA damage (33, 34, 35). Linking reduced fertility or increased incidences of reproductive developmental abnormalities with specific exposures and chemicals is particularly difficult in humans. Therefore, mechanisms underpinning the effects of gestational maternal smoking on the developing fetus remain very poorly understood.

Around half of the women having elective second-trimester terminations of normally progressing pregnancies smoke through the pregnancy. We therefore combined ongoing studies of fetal testis development (e.g. Ref. 36) with the in utero exposure of fetuses to cigarette smoke chemicals to address serious gaps in current knowledge of the effects of environmental chemicals on fetal human tissues and expression of fetal testicular genes and associated fetal endocrinology.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Study design

To test the hypothesis that maternal smoking affects the fetal testis in utero, all eligible women (see Sample collection) were recruited prospectively, with maternal and fetal morphological data recorded during consent and sample processing. In a subset of cases in which fetal cardiac blood was obtained, circulating hormones and cotinine were assayed and, in another subset of cases, the expression levels of key developmental genes were determined. Cases were matched for gestational stage between smoking and control (nonsmoking) groups.

Sample collection

The collection of fetal material (detailed in Ref. 36) was approved by the National Health Service Grampian Research Ethics Committees (REC 04/S0802/21). Women seeking elective, medical terminations of pregnancy were recruited with full written, informed consent by nurses working independently at Aberdeen Pregnancy Counseling Service. There was no change in patient treatment or care, and women were able to withdraw from the study at any point. Only normally progressing pregnancies (determined at ultrasound scan) from women older than 16 yr of age with a good grasp of English and between 11 and 21 wk of gestation were collected. Maternal morphological data and numbers of cigarettes smoked per day during pregnancy were recorded. Fetuses were transported to the laboratory within 30 min of delivery, weighed, crown-rump length recorded, and sexed. Where possible, an ex vivo blood sample was collected by cardiac puncture and the plasma stored at –20 C. The gonads were weighed and either snap frozen in liquid nitrogen and stored at –85 C or fixed for 5.5 h in Bouins, transferred to 70% ethanol, and processed for histology (37, 38).

The fetuses used for quantitative PCR (qPCR) and stereology are the same as those reported in the serial study of gene expression changes across the second trimester reported by O’Shaughnessy et al. (36). The present study focused on the effects of maternal smoking rather than expression changes across the second trimester and, in addition, also reports on the fetal testis gene expression of desert hedgehog (DHH), steroid acute regulatory protein, androgen receptor, and P450 oxidoreductase.

Endocrine and cotinine assays

Testosterone, a key marker of masculinization, was determined using a DELFIA kit (PerkinElmer Life Sciences, Cambridge, UK) with a detection limit of 0.3 nmol/liter and intra- and interassay coefficients of variation of 3.2 and 8.6%, respectively. Because of potential interference with the testosterone assay owing to the many hemolyzed samples, all 34 fetal plasma samples available were extracted on C18 spin columns (GE Healthcare Bucks, UK; C18 Self Pack POROS 10 R2; Applied Biosystems Ltd., Warrington, UK). Extracted samples (see supplemental data, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org) were reconstituted in assay buffer (PerkinElmer Life Sciences) and used for testosterone assay. The recovery of testosterone was 122 ± 9.7%. LH and intact human chorionic gonadotropin (hCG) were measured using DELFIA kits robust for hemolysis, with detection limits and inter- and intraassay coefficients of variation of 0.05 U LH/liter, 2.4 and 4.2%, and 0.5 U hCG per liter, 4.1 and 4.6%. There was a 0.02% cross-reaction of intact hCG in the LH assay. Cotinine, a metabolite of nicotine and a marker of smoking, was determined with a kit (Cozart Plc., Abingdon, Kent, UK) using a 25-ng cotinine per milliliter cutoff to maintain cross-reaction with nicotine at 0.6%. The determination was semiquantitative, based on calibrators at 0, 10, 25, and 50 ng cotinine per milliliter, with values between 0 and 12 ng cotinine per milliliter being considered negative.

Morphology, stereological determination of cell numbers, and immunohistochemistry

For immunohistochemistry 5-µm testis sections were stained with hematoxylin and eosin or mounted onto ChemMate slides (DakoCytomation Ltd., Ely, UK). Sections were dewaxed and endogenous peroxidase activity quenched (3% H2O2, PBS, 15 min). After blocking (PBS, 4% serum, 0.3% BSA, 2 h), primary antibody was added overnight at 4 C [anti-Dhh (N-17), 1:100 in blocking buffer; sc-33940; Santa Cruz Biotechnology, Santa Cruz, CA]. Slides were washed and secondary antibody (biotinylated rabbit antigoat IgG; B-7014; Sigma, Poole, UK. 1:200 in blocking buffer) added for 1 h, 22 C. After washing, color reaction was developed by 30 min incubation in Vectastain R.T.U. Elite ABC reagent (VectorLabs, Peterborough, UK) followed by 20 min in 800 µg/ml diaminobenzidine, 50 mM Tris (pH 7.6), 0.5% H2O2. Sections were washed in water and counterstained with hematoxylin. For stereology, testes were embedded in Technovit 7100 resin, cut into 20-µm sections, and stained with Harris’ hematoxylin. Total testis volumes were estimated [Cavalieri principle (39)] from the same slides used to determine cell number. Leydig, Sertoli, and germ cell numbers were counted using the optical dissector technique (40). Sertoli and germ cells were identified by their distinctive nuclei and position within the tubule (41, 42), whereas the Leydig cells were identified by their interstitial location, round nuclei, and prominent nucleoli (41, 42). Total germ cell number was measured without categorization as gonocytes, intermediate cells, or prespermatogonia (43, 44, 45). The numerical density of each cell type was estimated using an Olympus BX50 microscope fitted with a motorized stage (Prior Scientific Instruments, Cambridge, UK) and Stereologer software (Systems Planning Analysis, Alexandria, VA). Cross-sectional tubule diameters were measured (six fields of view per two sections per testis, x20) using an Olympus BX41 with a ProgRes C5 digital camera and ProgRes CapturePro (Jentopik Laser Optic Systeme GmbH, Jena, Germany).

Real-time qPCR

For quantification of 30 specific mRNA species in testes (see supplemental data), real-time PCR was used after reverse transcription of the isolated RNA. To allow specific mRNA levels to be expressed per testis and to control for RNA extraction efficiency, RNA degradation, and reverse transcription step, 5 ng external standard (luciferase mRNA; Promega UK, Southampton, UK) was added to each testis at the start of the RNA extraction (46). Testis RNA was extracted using TRIzol (Life Technologies, Paisley, UK), and the RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies) (47). The real-time PCR approach used the SYBR green method in a 96-well plate format using a MX3000 cycler (Stratagene, Amsterdam, The Netherlands). Reactions contained 5 µl 2x SYBR master mix (Stratagene), primer (100 nM), and template in a total volume of 10 µl. At the end of the amplification phase, a melting curve analysis was carried out on the products formed. All primers were designed by Primer Express 2.0 (Applied Biosystems) using parameters previously described (46).

PAH determinations

The fetal liver concentrations of 16 PAHs known to be in cigarette smoke (e.g. Ref. 48) were determined. Seven internal standards (see supplemental data) were added at 0.05 µg to freeze-dried livers and PAHs extracted into ethanoic potassium hydroxide (90 C, 8 h) followed by 3 x 10 ml isohexane and evaporation to 3 ml. After absorption chromatography to remove lipids, samples were loaded onto 10-g silica columns (Merck, Nottingham, UK) previously conditioned with 40 ml isohexane (Rathburn Chemicals, Walkerburn, UK). Fractions were eluted with 75 nM isohexane/dichloromethane (1:1 vol/vol) and concentrated under nitrogen. PAH content was determined using gas chromatography linked to mass spectrometry operated in the single ion monitoring mode (Trace MS; Thermo Electron, Hemel Hempstead, UK) linked to a Trace 2000 GC fitted with an AS2000 autosampler). Separations were effected on a Zebron ZB5 fused silica capillary column coated with 95% dimethylpolysiloxane/5% phenyl with a phase thickness of 0.25 mm (Phenomenex, Macclesfield, UK). The operating temperature for each PAH analysis started at 70 C for 3 min, was then ramped at 5 C/min to 250 C and held for 1 min, ramped to 300 C at 6 C/min and held for 6 min, and then ramped to 325 C at 10 C/min and held for 5 min. The carrier gas was helium and samples were injected in splitless mode with a surge. The mass spectrometer was operated in the electron ionization mode at 70 electron volts and a source temperature of 250 C. The ions monitored for each compound were between 128 and 278 m/z (see supplementary data). Response factors were calculated relative to the seven internal standards. Certified reference materials could not be found for sample matrix under investigation, and reproducibility was therefore monitored by repeated analysis of spiked liver tissue. The recovery values for PAH analysis were between 81 and 116% (see supplementary data).

Statistical analysis

Analyses were performed using JMP (5.1; Thompson Learning, London, UK) and Minitab (14.12; Minitab UK Ltd., Coventry, UK). Normality of data distribution was tested with the Shapiro-Wilk test and nonnormally distributed data were log transformed and rechecked for normality before analysis. Care was taken to ensure that no comparison involved groups with any difference between smokers and nonsmokers in terms of stage of gestation, thus avoiding bias. Because most of the genes investigated showed changes in expression across the second trimester (36), two-way ANOVA was used to test the combined effects of gestational age (weeks) and maternal smoking (yes/no) on morphological and biochemical data and gene expression levels. To ensure that statistically significant differences were robust, these analyses were repeated with the weeks of gestation divided into 11–13, 14–16, and 17–19 wk and less than 16 and more than 16 wk, and the statistical findings were the same. Relationships between variables were explored by linear discriminant analysis to classify observations, with a cross-validation procedure to avoid bias. We assessed the importance of genes and PAHs in their ability to classify mothers into smoking or nonsmoking categories via a simple test of a binomial proportion on the number of patients classified correctly; the null hypothesis of no information corresponds to a classification probability of one half for the binomial proportion, enabling us to obtain a P value for the actual number classified correctly. After initial data analysis, the DHH qPCR was repeated twice to confirm the findings, and the results for DHH are expressed as a mean of three separate determinations for each testis. For the cotinine assay, values between 0 and 50 ng/ml were expressed at the detected values, whereas all values less than 0 ng/ml were expressed as 0 ng/ml and values greater than 50 ng/ml were expressed as 51 ng/ml for the purposes of statistical analysis.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Table 1Go summarizes our morphological and endocrine data. The only significant differences in the maternal smoking group were reduced fetal intact hCG (maternal in origin) and elevated fetal cotinine. Only fetuses from mothers who stated that they continued to smoke during gestation had circulating cotinine values greater than 12 ng/ml, confirming that metabolites of nicotine had reached the fetal circulation. Fetal cotinine levels did not correlate significantly with indices of fetal growth including length (P = 0.596), weight (P = 0.835), or testis weight (P = 0.842). Comparing fetuses from nonsmoking with smoking mothers, there were also no significant differences in testicular indices, including testis volume (11.4 ± 1.4 vs. 12.7 ± 1.1 mm3, P = 0.813), cord diameter (113 ± 6 vs. 125 ± 6 µm, P = 0.273), Sertoli (3.5 ± 0.8 x 106 vs. 3.8 ± 0.6 x 106, P = 0.978), Leydig (1.0 ± 0.2 x 106 vs. 1.28 ± 0.1 x 106, P = 0.290), or germ (0.6 ± 0.1 x 106 vs. 0.6 ± 0.1 x 106, P = 0.920) cell numbers. Fetal testosterone was slightly, but not significantly, reduced in fetuses from mothers who smoked. The LH+hCG to testosterone ratio was calculated as an index of testicular responsiveness to LH receptor activation. Although the ratio was increased 1.6-fold in fetuses whose mothers smoked (13.7 ± 2.9 vs. 21.8 ± 3.8, P = 0.172), suggesting some reduction in testicular responsiveness to LH/hCG, this was not statistically significant.


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  		TABLE 1. Morphological and endocrine data for mothers and male fetuses (mean ± SEM)

 
Fetal testicular mRNA transcript levels (Table 2Go) were only significantly altered by maternal smoking in the case of DHH. Of the remaining 29 genes, and based on 20% or greater change, seven (CYP11B1, PRLR, SOX9, WNT2B, SF1, CXORF6, GRK2) showed a downward trend if the mother smoked, six (AHR, LHR, MC2R, CYP21, KIT, FGF9) showed an upward trend, and the remaining 16 showed no trend with respect to maternal smoking. DHH increased significantly across the second trimester (P < 0.001), and by 19 wk, the product was clearly localized to Sertoli cells (Fig. 1Go), with lighter staining at 13 and 15 wk (not shown). Discriminant analysis of mRNA transcript levels and fetal or maternal indices, such as fetal crown-rump length, achieved 95.9% (P < 0.001) correct classification of fetuses within smoking or nonsmoking (control) groups using the combination of PTC1, FGF9, WT1, GATA4, CYP11B1, and GRK2 as discriminators, with PTC1 emerging as the most significant single discriminator. This high classification rate was not merely a chance result; many other combinations of genes involving these genes or subsets of these genes produced success rates as high or almost as high (but still significant).


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  		TABLE 2. Effects of maternal smoking on the mRNA transcript levels of 30 key genes in the human male fetal testis (mean ± SEM)

 

Figure 1
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  		FIG. 1. Fetal human testicular DHH protein is predominantly located in the Sertoli cells. DHH was localized predominantly to the Sertoli cells in the seminiferous tubules (ST). Some light staining was observed in the Leydig cell containing interstitial area. Representative section from a 19-wk-old fetus.

 
Fetal liver concentrations of 14 PAHs showed no significant differences between fetuses from mothers who smoked or not during gestation (Table 3Go), although there was marginal significance in the case of benzo[a]anthracene. In terms of a 20% or greater change if the mother smoked, seven showed a downward trend, three showed an upward trend, and the remaining four showed no trend. Fetal liver concentrations of four PAHs changed significantly during the second trimester (comparing ≤ 16 wk vs. > 16 wk), acenaphthene (P = 0.006), and benzo[a]anthracene (P < 0.001) fell 2.2- and 5.5-fold, respectively, whereas benzo[b]fluoranthene (P = 0.007) and anthracene (P = 0.006) rose 9.7- and 2.3-fold, respectively. In the case of chrysene, there was a significant interaction, with maternal smoking affecting the way in which fetal liver titers changed (P = 0.030). Using discriminant analysis (Fig. 2Go) to tease out complex changes in PAHs showed that combining anthracene, acenaphthene, benzo[a]pyrene, and fluorine as discriminators correctly classified 100% of samples (P < 0.001).


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  		TABLE 3. Concentrations of selected PAHs in the human male fetal liver (mean ± SEM)

 

Figure 2
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  		FIG. 2. Fetal human PAH concentrations are predictors of maternal smoking. Canonical plot of representative PAHs used in discriminant analysis against presence or absence of maternal smoking. Using anthracene, acenaphthene, BaP, and fluorine (in bold) as discriminators allocated 100% of fetal livers to the correct subset (P < 0.001). IcdP, Indeno[1,2,3-cd]pyrene; BaA benzo[a]anthracene; BkF, benzo[k]fluoranthene; BbF, benzo[b]fluoranthene; DahA, dibenzo[a,h]anthracene; BaP, benzo[a]pyrene.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have shown that in utero exposure of the developing human fetus to a complex mix of chemicals in cigarette smoke can alter testicular gene expression before there are any obvious signs of growth retardation. The reduction in second-trimester testicular DHH expression is the first report of a specific mechanism that may link gestational maternal smoking with strong epidemiological data. This effect was not associated with any large changes in fetal liver PAH titers.

In this study we clearly demonstrated, through discriminant analysis, that subtle combinations of fetal liver PAHs can correctly classify the fetus into smoking or nonsmoking groups. Thus, whereas smoking induced no marked, significant changes in fetal liver PAHs, there are subtle changes that may have effects on fetal development. The fact that we observed no significant alterations in any individual fetal liver PAHs is important but not surprising because many PAHs are not markedly increased in tissues from smokers. For instance, of 17 assorted PAHs measured in two studies of adult lungs (49, 50), the former reported only the antiandrogenic dibenzo[a,h]anthracene as significantly higher, whereas the latter reported five (including BaP) significantly higher, in smokers’ lungs. Although not significantly altered if the mother smoked, dibenzo[a,h]anthracene was up to 4 times higher in the fetal human liver than reported values for adult smoker’s lungs. We found no report of fetal human tissue levels of PAHs, but the other PAHs reported in adult lungs were all at least 50% lower than the levels we observed in the fetal human livers. There is therefore significant fetal liver accumulation of PAHs and other environmental toxins such as polybrominated diphenyl ether (51), which also cross into the fetus. The mechanisms by which toxins arising from maternal gestational smoking can affect reproductive development and fertility of male offspring remain unclear, although many cigarette smoke chemicals can act via the aryl hydrocarbon receptor (AhR) (52, 53), which is expressed in the germ cells of the human fetal testis (54). A similar effect is seen in the female in which AhR activation is important in determining the eventual size of the ovarian reserve (55). Exposure to PAHs and other AhR ligands such as dioxin induces oocyte loss, in part at least, by increasing proapoptotic bax expression (56, 57). The fact that we observed no effect of maternal smoking on fetal testis germ cell numbers during the second trimester suggests that any effect of in utero exposure on eventual spermatogenesis may therefore manifest later in gestation.

Our observation that DHH expression is reduced in fetuses whose mothers smoked is important and is consistent with published epidemiological data on the effects of gestational maternal smoking on sons (6, 17). Men with DHH mutations are subfertile, and in both humans and mice, the phenotypic effects of defective DHH signaling vary, depending on the individual’s genetic background (58, 59). This suggests important dosage effects of DHH gene expression, but no data for this are available in the literature. An example of the potential importance of this is the dosage of the transcription factors Friends of GATA-2 and GATA4, which are haploinsufficient in testis development (60). Given that both the DHH knockout mouse and DHH mutant men show extreme deviation from normal testis development, it is entirely possible that a reduction in DHH signaling would be sufficient to cause the much more subtle defects related to maternal gestational smoking and reported by many studies. The DHH signaling system is known to be vulnerable to direct estrogenic regulation in the uterus (61). It is therefore reasonable to hypothesize that inappropriate endocrine signaling, resulting from exposure to PAHs, and other cigarette smoke chemicals may be able to disturb DHH in the fetal testis. In addition, combined expression of DHH and steroidogenic factor 1 (SF1) is required for normal Leydig cell development (62), which makes our finding of a trend toward reduced SF1 in association with gestational smoking a further indication of potential abnormalities in Leydig cell development. The cognate receptor for DHH is patched 1 (PTC1) and normally DHH/PTC1 signaling triggers differentiation of precursor Leydig cells rather than affecting interstitial Leydig cell proliferation or survival (63). Because up-regulation of PTC1 is a readout of effective hedgehog signaling (64, 65), it is pertinent that PTC1, which tended to be lower in smokers, was the most important gene in correctly categorizing fetuses to smoking or nonsmoking groups (removal of this gene from the discriminant analysis resulted in a much reduced success rate). In addition, whereas there was no statistically significant effect of maternal smoking on testosterone, fetal testosterone titers tended to be lower if the mother smoked. This suggests that maternal smoking acts subtly on a range of cells and signaling systems. We propose that chemicals, including PAHs, from maternal intake of cigarette smoke act to reduce DHH/PTC1 signaling, reducing the up-regulation of SF-1 and thus lowering the number of precursor cells for the second generation of Leydig cells, possibly reducing testosterone later in fetal and neonatal development. That continued exposure to PAHs impairs human sexual maturation (66) also suggests complex and ongoing effects of impaired DHH signaling, which remains important in the adult testis (67).

It is reasonable to conclude that the effects on gene expression we observed are specific and not simply due to reduced fetal growth because intrauterine growth retardation alone does not compromise pituitary-testis function (68). Some older studies (e.g. Ref. 69) reported that birth weight, and therefore reduced in utero growth, is an important determinant of hypospadias and cryptorchidism, which suggests that a combined effect of a wide range of sequelae to maternal cigarette smoke may underpin the observed phenotypic effects. Indeed fetal biparietal diameter is reduced as early as 20–24 wk by maternal smoking during gestation (70). This is later than in our study but pertinent for the development of the fetal ovary given reduced fertility in women exposed to cigarette smoke in utero (71, 72). Interestingly, a woman’s in utero exposure to her mother’s cigarette smoke appears to affect her own offsprings’ birth weight (73), indicating complex and cumulative effects of in utero exposure to complex cocktails of chemicals.

Given the absence of second-trimester effects of maternal smoking on fetal or testis size, key testis cell numbers, circulating testosterone, and LH concentrations or the expression of 29 key developmental genes, we conclude that the observed reduction in DHH may be part of a mechanism linking maternal gestational smoking with an increased incidence of impaired reproductive development in male offspring.


   Acknowledgments
 
The impartial help of the staff of the Pregnancy Counseling Service was essential for the collection of the fetuses. We are grateful to Ms. Margaret Fraser and Gillian Moir and the staff of the Histology and Electron Microscopy Facility (Institute of Medical Sciences, University of Aberdeen) for their expert technical assistance. We thank Claire Mackie (Macaulay Institute) for the PAH analyses. We are grateful to the Society for Reproduction and Fertility for the summer vacation scholarship that supported S.C.


   Footnotes
 
This work was partly supported by grants from the Chief Scientist Office (Scottish Executive), the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust. M.J.B. was funded by the Scottish Executive.

Disclosure Information: All authors have nothing to declare.

First Published Online November 13, 2007

Abbreviations: AhR, Aryl hydrocarbon receptor; DHH, desert hedgehog; hCG, human chorionic gonadotropin; PAH, polycyclic aromatic hydrocarbon; PTC1, patched 1; qPCR, quantitative PCR; SF1, steroidogenic factor 1.

Received August 20, 2007.

Accepted November 7, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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