Mol. Hum. Reprod. Advance Access originally published online on January 23, 2007
Molecular Human Reproduction 2007 13(4):223-229; doi:10.1093/molehr/gal114
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Modifying effect of the AR gene trinucleotide repeats and SNPs in the AHR and AHRR genes on the association between persistent organohalogen pollutant exposure and human sperm Y : X ratio
1 Department of Clinical Sciences, Molecular Reproductive Medicine Research Unit 2 Fertility Centre, Scanian Andrology Centre, Malmö University Hospital, Lund University, Malmö, Sweden 3 Division of Occupational and Environmental Medicine and Psychiatric Epidemiology, Lund University Hospital, Lund, Sweden
4 To whom correspondence should be addressed at: Department of Clinical Sciences, Molecular Reproductive Medicine Research Unit, Malmö University Hospital, CRC, Building 91, Plan 10, Entrance 72, SE 205 02 Malmö, Sweden. E-mail: tarmo.tiido{at}med.lu.se
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Abstract |
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Persistent organohalogen pollutants (POPs) have been suggested to be involved in changing the proportion of ejaculated Y-bearing sperm. The androgen receptor (AR), aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor repressor (AHRR) may modulate the effect of POPs with regard to previously observed sperm Y : X ratio changes. The objective of this study was to investigate whether sperm Y : X ratio changes in subjects exposed to 2,2'4,4'5,5'-hexachlorobiphenyl (CB-153) and dichlorodiphenyl dichloroethene (p,p'-DDE) were modified by polymorphisms in the AR, AHR and AHRR genes. Semen for analysis of Y- and X-bearing sperm by two-colour fluorescence in situ hybridization and blood for leukocyte DNA genotyping and analysis of CB-153 and p,p'-DDE concentrations were obtained from 195 Swedish fishermen. The polymorphic CAG and GGN repeats in the AR and the R554K and P185A single-nucleotide polymorphisms in the AHR and AHRR genes, respectively, were determined by direct sequencing and allele-specific PCR. The effect of p,p'-DDE was modified by CAG or GGN repeat category in relation to the proportion of Y-bearing sperm (P = 0.005 and 0.02 for CAG and GGN, respectively). Moreover, p,p'-DDE, but not CB-153, levels were associated with Y-sperm proportion in men with CAG < 22 (P < 0.001), but not in those carrying CAG
22 (P = 0.73). This association was even more pronounced in subjects carrying a short CAG repeat in combination with an AHRR G-allele. The association in regard to p,p'-DDE was found for GGN = 23 but not for the GGN < 23 or GGN > 23 subgroups (P = 0.01, 0.44 and 0.99, respectively). In conclusion The endocrine-disrupting action of POPs, in relation to the observed changes in sperm Y : X ratio, may be modulated by the genes involved in sex steroid and dioxin-mediated pathways. Key words: androgen receptor/aryl hydrocarbon receptor/polymorphisms/POPs/sperm Y: X ratio
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Introduction |
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Persistent organohalogen pollutants (POPs), e.g. polychlorinated dibenzofurans, polychlorinated dibenzo-p-dioxins, polychlorinated biphenyls (PCBs), dichlorodiphenyl trichloroethane (DDT) and dichlorodiphenyl dichloroethene (p,p'-DDE), the most stable metabolite of DDT, are widespread in the environment, potent toxicants and resistant to metabolic breakdown, leading to accumulation of these compounds in the food chain. A number of POPs can cause adverse effects in the endocrine system and induce a wide range of toxic responses (Toppari et al., 1996).
Recent studies have indicated that the proportion of new-born boys has been declining in many countries during the past five decades (Allan et al., 1997; van der Pal-de Bruin et al., 1997; Marcus et al., 1998; Møller, 1998; Parazzini et al., 1998) and the exposure to POPs or other environmental toxins at the time of testicular development is thought to represent one important cause of such a time-related trend (Toppari et al., 1996; Skakkebaek et al., 2001).
Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Seveso, Italy was associated with a subsequent decrease in the proportion of male offspring of men who were exposed in adolescence or earlier in life (Mocarelli et al., 1996, 2000; del Rio Gomez et al., 2002). In human populations exposed to more moderate levels of POPs, both increased (Karmaus et al., 2002) and decreased (Rylander et al., 1995) sex ratios (male:female proportions) have been reported. Recently, it was shown that in utero and lactational exposure of male rats to dioxin decreased the sex ratio of the subsequent generation (Ikeda et al., 2005).
The offspring sex ratio changes resulting from the POP exposure may be related to a skewed proportion of Y- and X-chromosome-bearing sperm. We have recently reported a moderate positive association between serum levels of the POP biomarkers 2,2'4,4'5,5'-hexachlorobiphenyl (CB-153) and p,p'-DDE and the proportion of Y-bearing spermatozoa in a population comprising Swedish fishermen (Tiido et al., 2005). These fishermen constitute a population with high fish consumption and have previously been found to eat, on an average, more than twice as much locally caught fatty fish than the subjects from the general Swedish population, which has resulted in increased POP levels in plasma among fishermen (Svensson et al., 1995).
Much of the evidence from various disciplines has been accumulated in respect to both animal and human data to support the influence of androgens and estrogens in regulation of sex ratio but no experimental evidence exists to date to confirm hormonal determination of sex ratio (James, 1996, 2001). Therefore, it is possible that the sex-hormone-dependent biological responses leading to sex ratio changes may occur on account of agonistic or antagonistic action of POPs as ligands for sex hormone receptors (Kuiper et al., 1998) and through aryl hydrocarbon receptor (AHR)-mediated interference of androgen receptor (AR) and/or estrogen receptor (ER) signalling (Jana et al., 1999; Klinge et al., 1999). In addition, POPs may affect hormone concentrations in men moderately exposed to p,p'-DDE and CB-153 as previously reported (Rylander et al., 2006).
The vast majority of the toxic effects of dioxins, dibenzofurans and PCBs are mediated through the AHR, which is a ligand-activated transcription factor (Mimura and Fujii-Kuriyama, 2003). Upon ligand binding, the AHR translocates from the cytoplasm to the nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), binds to xenobiotic response enhancer elements and activates the transcription of target genes such as cytochrome P450 1A1 (CYP1A1). CYP1A1 is a protein involved in the metabolism of a large number of xenobiotics and also plays an important role in estrogen metabolism. The aryl hydrocarbon receptor repressor (AHRR) inhibits the AHR and constitutes a negative feedback loop in the dioxin-related signal transduction pathway (Mimura and Fujii-Kuriyama, 2003). It is plausible to believe that variations in any of these genes could influence the susceptibility to dioxins.
The most widely studied functional single-nucleotide polymorphism (SNP) in the human AHR gene is G > A substitution in exon 10, which causes an arginine-to-lysine change (R554K) in the transactivating domain (Harper et al., 2002). The only known polymorphism in the human AHRR gene is a C > G change in exon 5, which encodes a proline-to-alanine substitution at codon 185 (P185A) (Harper et al., 2002). In the Japanese population, P185A has been linked to the risk of developing micropenis (Fujita et al., 2002; Soneda et al., 2005), the susceptibility and severity of endometriosis (Tsuchiya et al., 2005) and defective spermatogenesis in infertile men (Watanabe et al., 2004).
Androgen action on seminiferous tubules is essential for full, quantitatively normal spermatogenesis and fertility. This effect is achieved by androgen action on Sertoli cells, mediated by the AR (De Gendt et al., 2004). The N-terminal-transactivating domain of the AR gene is highly polymorphic because of two amino-acid stretches encoded by trinucleotide repeat tracts of variable length, (CAG)n CAA coding for glutamine residues and, further downstream, (GGT)3GGG(GGT)2 (GGC)n (the GGN repeat) encoding glycines. The significance of the CAG and GGN repeats for the transactivating potential of the receptor has been shown in vivo (La Spada et al., 1992; Aschim et al., 2004) as well as in vitro (Gao et al., 1996; Tut et al., 1997), underlining the importance of these repeats for optimal receptor function. Recently, we have reported that the negative association between p,p'-DDE serum levels and sperm number as well as chromatin integrity is limited to subjects with a CAG repeat length of 
21 (Giwercman et al., 2006). These findings indicated a gene–environment interaction in relation to impairment of male reproductive function in response to POP exposure.
Keeping our previous findings regarding the effect of POP exposure on sperm Y : X chromosome ratio in mind, we investigated which roles the AR polymorphisms as well as the R554K and P185A in the AHR and AHRR genes, respectively, may play in modifying the effect of exposure to CB-153 and p,p'-DDE in regard to sperm Y : X ratio.
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Subjects and methods |
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Study population
The original cohorts consisted of Swedish fishermen and have in detail been described previously (Svensson et al., 1995). In the year 2000, a postal questionnaire, mainly focusing on fracture incidence, but also including a question concerning interest in more information on a study of male semen function, was sent to 5183 fishermen from the original cohorts, born 1935 or later. Out of 2614 subjects, who responded to this specific question, 479 wanted more information about the semen study. After further information, 266 men agreed to participate. During the study period, 71 subjects were excluded because of logistical reasons, change of mind, sickness or recent vasectomy.
The non-participants from the original fishermen's cohort had a similar age distribution as the participants (Rignell-Hydbom et al., 2004). Circumstantial evidence based on data from the Swedish Medical Birth Register supports that there was no difference in number of fathered children between participants and non-participants.
Out of 195 men who finally participated in the semen study, 183 donated enough semen for fluorescence in situ hybridization (FISH) analyses. Out of these, 28 samples were excluded because of a low number of cells available or failure during analysis. There were no statistical differences with respect to age, lipid-adjusted levels of CB-153 and p,p'-DDE, proportion of progressively motile sperms, sperm concentration or total sperm count, between the 155 men included in the present study and the 28 subjects who were excluded (21 subjects with hybridization failure and 7 with low sperm number). Exposure data were lacking in 6 men and blood samples for DNA analysis in 10 men. Additionally, genotyping was unsuccessful in one subject, four subjects and one subject for AR GGN repeats or AHR R554K and AHRR P185A variants, respectively. Therefore, the final results are based on a total of 143 subjects for AR CAG, 142 subjects for AR GGN, 139 subjects for AHR R554K and 142 subjects for AHRR P185A variants.
Collection of biological material and questionnaire data
A mobile laboratory unit was established for collection and analysis of semen and blood samples (Rignell-Hydbom et al., 2004). Information on how to collect the semen sample was given to the participants during a telephone interview as well as in written form. The subjects were informed to keep an abstinence period of 3–4 days before collection and in each case the actual length of abstinence period was recorded (median 3.0, range 0.5–15). Venous blood samples were drawn between 7 am and 9 pm and were used for genotyping of DNA polymorphisms. Furthermore, the samples were centrifuged and sera were frozen at –80°C for later analysis of exposure biomarkers.
All men who participated gave their written informed consent and the study was approved by the Ethics Committee of Lund University.
Two-colour FISH and scoring criteria
Preparation of sperm, in situ hybridization and determination of scoring criteria were essentially as described in Tiido et al. (2005). Briefly, sperm heads on slides were decondensed and hybridized with fluorescent probes. Thereafter, the microscopic examination with an Olympus AX 70 epifluorescence microscope was performed blindly, i.e. without knowledge of the exposure levels or other subject characteristics. The X-specific probe emitted a red signal and the Y chromosome specific probe a green signal. In every sample, the proportion of sperm showing a clear red or green signal was 95%.
The presence of Y/X chromosome in the spermatozoa was evaluated by assessing randomly selected visual fields. Inter- and intra-observer coefficients of variation with respect to the proportion of Y-bearing sperms were estimated to be 2.3% and 3.3%, respectively, by scoring 500 cells only and this procedure was subsequently aimed to be applied. However, because of the quality of the slides in six samples, the number of nuclei scored was <500 (4% <500).
Determination of CB-153 and p,p'-DDE in serum
Analyses of CB-153 and p,p'-DDE in serum were performed by applying solid-phase extraction using on-column degradation of the lipids and analysis by gas chromatography mass spectrometry as previously described (Richthoff et al., 2003; Rignell-Hydbom et al., 2004). Levels of detection, CV and participation in the quality-control programmes have been described in detail elsewhere (Jönsson et al., 2005).
Determination of lipids by enzymatic methods
Plasma concentrations of triglycerides, cholesterol and phospholipids were determined by enzymatic methods using reagents from Boehringer–Mannheim (triglycerides and cholesterol; Mannheim, Germany) and Waco Chemicals (phospholipids; Neuss, Germany). The total lipid concentration in plasma was calculated by summation of the amounts of triglycerides, cholesterol and phospholipids. In these calculations, the average molecular weights of triglycerides and phospholipids were assumed to be 807 and 714. For cholesterol, we used an average molecular weight of 571, assuming that the proportion of free and esterified cholesterol in plasma was 1 : 2.
Analysis of CAG and GGN repeat lengths in the AR gene
Analyses of AR gene polymorphisms were performed according to Lundin et al. (2006). Briefly, genomic DNA was prepared from peripheral leukocytes, and the CAG and GGN repeats were amplified by PCR for 40 cycles in a 50 µl incubation mixture containing 50 ng DNA as well as 0.5 and 0.3 µmol l–1 of flanking primers for CAG and GGN repeats, respectively. Each amplification cycle included denaturation at 96°C for 1 min or 45 s for CAG and GGN repeats, respectively, and primer annealing for 45 s at 61°C for both the CAG and GGN repeats. Primer extension occurred at 72°C for 1 min, with an initial denaturation step at 96°C for 3 min and a final extension step at 72°C for 7 min. The PCR products were purified using JETQUICK PCR purification kit (Genomed GmbH, Bad Oeynhausen, Germany) according to protocol provided by the manufacturer. Approximately 30 ng of the purified products were used in a sequencing reaction with the BigDye® Terminator v3.1Cycle Sequencing kit (Applied Biosystems, Foster City, USA). Samples were analysed externally on an Applied Biosystems Model 3730 Automated Capillary DNA Sequencer (Applied Biosystems).
Analysis of polymorphisms in the AHR and AHRR genes
The polymorphic loci of AHR and AHRR genes were genotyped by allele-specific PCR (AS-PCR). For each polymorphism, two reactions per subject were run, using a specific primer for each variant together with an upstream and a downstream primer. PCR conditions were established to generate both a control fragment and a shorter, allele-specific band in the presence of the variant and only the control fragment in its absence. AS-PCR of the AHR R554K polymorphism was performed in a total volume of 50 µl containing 50 ng genomic DNA, 45 mmol l–1 KCl, 10 mmol l–1 Tris HCl (pH 9.1), 200 µmol l–1 deoxynucleotide triphosphate, 1.5 mmol l–1 MgCl2, 0.5 U Dynazyme Taq polymerase (Finnzymes Oy, Espoo, Finland), 0.5 µmol l–1 of each of the primers AHR554 forward (Fw), AHR554 reverse (Rev) and 0.5 µmol l–1 of either AHR554 RevG or AHR554 RevA. Primer sequences are presented in Table I. Amplification was performed for 32 cycles; each cycle included denaturation for 1 min at 96°C, primer annealing for 30 s at 55°C, and primer extension for 1 min at 72°C, with an initial denaturation step for 3 min at 96°C, and a final extension step for 7 min at 72°C. Regarding the AHRR P185A polymorphism, an annealing temperature of 57°C for 30 s was used, and the PCR amplification was performed for 35 cycles. Other conditions were the same as for the AHR R554K reaction. The sequences of the primers are presented in Table I. The control fragment and the allele-specific fragment were 504 and 206 bp, respectively, for the AHR R554K polymorphism and 498 and 331 bp, respectively, for the AHRR P185A polymorphism (Figure 1). One sample of each different genotype was sequenced to confirm the expected sequences.
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Statistical analysis
In linear-regression models, we evaluated whether CAG and GGN repeat length and SNPs for AHR and AHRR modified the impact of CB-153 and p,p'-DDE exposure on the fraction of Y–chromosomes. CAG repeat numbers were categorized into two categories of almost equal size: <22 and
22. GGN lengths were divided into three categories: the most common length of 23; <23 and >23. The C allele of AHRR P185A polymorphism has been suggested to increase predisposition to micropenis and male infertility, mainly in the C/C homozygotes (Watanabe et al., 2004; Soneda et al., 2005). In accordance with these studies, the subjects were classified in two categories: C/C and C/G combined with G/G. Regarding AHR R554K, we categorized genotypes into two categories as G/G and G/A combined with A/A, respectively, because the A/A group comprised <2% of all subjects. Since the CB-153 and p,p'-DDE serum levels were highly correlated (r = 0.73), both variables were not simultaneously taken into the models. Initially, these POP variables were analysed as continuous variables (untransformed and log transformed). Model assumptions were checked by means of residual analyses which pointed to no need for log transformation of the exposure parameters in order to achieve a normal distribution of the residuals. On the basis of the former studies on this population (Tiido et al., 2005, 2006), age (continuous) and abstinence time (continuous) were always included as confounders.
Primarily, for each genetic polymorphism, we tested for difference in the proportion of Y-sperms between the genotype groups. The potential of CAG or GGN number and genotype for AHR and AHRR as modifiers of the effect of exposure was investigated by including interaction terms in the regression models (i.e. CAG <22/22 * CB-153/p,p'-DDE; GGN < 23/23/>23 * CB-153/ p,p'-DDE; AHR category * CB-153/p,p'-DDE; and AHRR category * CB-153/p,p'-DDE). Furthermore, the interaction between the AHR or AHRR genotype and AR polymorphisms (CAG or GGN) as modifiers of the effect of CB-153/p,p'-DDE exposure on the proportion of Y-sperm was evaluated by introducing the following variable: CAG <22/22/GGN < 23/23/>23 * AHR/AHRR category * CB-153/p,p'-DDE. In order to elucidate to which genotype the association between exposure and proportion of Y-sperm was limited, this association was subsequently tested for each genetic subgroup with CB-153 and p,p'-DDE as continuous variables.
When interaction term for continuous exposure variables was shown to be statistically significant, additional analyses with categorized exposure levels (three groups of equal size designated as low, medium and high) in regression models were accomplished.
All analyses were carried out by the SPSS software (SPSS for Windows 12.0; SPSS Inc., Chicago, IL, USA).
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Results |
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Distribution of the AR gene triplets and polymorphisms at AHR R554K and AHRR P185A
The number of AR CAG triplets varied between 17 and 30 with a median length of 22.0. The number of GGN triplets varied between 10 and 25. Among the study subjects, GGN = 23 was the dominating allele, comprising 51% (n = 73) of the whole study population (n = 142). The frequency of GGN = 24 was 28% (n = 39), whereas 15% (n = 21) had GGN < 23 and 6% (n = 9) GGN > 24. The genotype and allele frequencies of the AHR R554K and AHRR P185A polymorphisms are shown in Table II.
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Association between Y-sperm proportion and AR, AHR and AHRR genotypes
As shown in Table II, the mean Y-sperm frequencies were similar among subgroups divided according to CAG (<22;
22) and GGN (<23; 23; >23) length of the AR gene. Moreover, we did not find any difference between AHR R554K and AHRR P185A genotypes with regard to the mean proportion of Y-bearing sperm (Table II).
Interaction between exposure and AR
A statistically significant interaction was found between p,p'-DDE and CAG repeat category and between p,p'-DDE and GGN repeat length in relation to the proportion of Y-sperm (P = 0.005 and 0.02, respectively) (Table III). In the subgroup of men with CAG < 22 but not in the subgroup CAG 22, p,p'-DDE levels were significantly associated with the proportion of Y-sperm (P < 0.001 and 0.73, respectively), whereas in the case of CB-153, a trend toward significance was observed (P = 0.08 and 0.94, respectively). As shown in Table IV, the men with low p,p'-DDE exposure and CAG < 22 had somewhat lower proportion of Y-sperm when compared with the men with low p,p'-DDE exposure and CAG 22, but the situation was in the opposite direction looking at the men in the highest exposed p,p'-DDE group. Similarly, an association with regard to p,p'-DDE was found for GGN = 23 but not for the GGN < 23 or GGN > 23 subgroups (P = 0.01, 0.44 and 0.99, respectively).
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Interaction between exposure and AHR/AHRR
Despite the lack of statistically significant interaction between exposure and AHR (P = 0.92 for CB-153 and P = 0.88 for p,p'-DDE) or AHRR (P = 0.91 for CB-153 and P = 0.09 for p,p'-DDE) genotype, there was a difference in association between the p,p'-DDE levels and Y-sperm proportion between the AHRR P185A genotypes C/C and C/G + G/G (P = 0.86 and 0.01, respectively) (Table III).
AHRR–AR–exposure interaction
The interaction of p,p'-DDE levels with the AHRR genotype and the CAG group in relation to the proportion of Y-sperm was statistically significant (P = 0.02), whereas this was not true with CB-153 in the interaction model (P = 0.23). The interaction was not statistically significant (P = 0.62 for CB-153 and P = 0.14 for p,p'-DDE) either when the GGN category was combined with the AHRR genotype or when the CAG (P = 0.63 for CB-153 and P = 0.05 for p,p'-DDE) or GGN (P = 0.75 for CB-153 and P = 0.09 for p,p'-DDE) category was combined with the AHR genotype in the interaction model.
For p,p'-DDE, in a model of joint genotype data, the association with Y-sperm proportion in the group of CAG < 22 combined with AHRR P185A C/G + G/G was found to be statistically significant (ß = 1.43, P < 0.001), whereas this was not true for the remaining subjects—men with CAG < 22 and AHRR P185A C/C or those with CAG 22 combined with any of the AHRR P185A genotypes (ß = 0.41, P = 0.15) (Figure 2).
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When the p,p'-DDE level was not treated as a continuous variable, but divided into three (low, medium and high) categories (Table IV), none of the interactions between single or combined genotypes and the exposure level (see above) was statistically significant (all P > 0.1; data not shown). However, the span in the proportion of Y-sperm between the lowest and the highest exposure groups was more pronounced for CAG < 22, GGN = 23 and AHRR P185A C/G + G/G and for the combination of CAG < 22 and AHRR P185A C/G + G/G than in the complementary groups, thus showing the same trend as the calculations based on exposure as a continuous variable.
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Discussion |
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To investigate whether the previously observed sperm Y : X chromosome ratio changes in respect of exposure to CB-153 and p,p'-DDE were dependent on AR CAG and GGN repeat lengths as well as AHR R554K and AHRR P185A variants, DNA genotyping of Swedish fishermen was conducted. Interactions were found between p,p'-DDE and CAG repeat category and between p,p'-DDE and GGN repeat in relation to the proportion of Y-sperm. In the subgroup of men with CAG < 22 and GGN = 23, p,p'-DDE levels significantly associated with the proportion of Y-sperm. Thus, short CAG repeats and GGN = 23, which is the most common GGN length among Caucasians, seem to modify the association between p,p'-DDE and Y-sperm proportion. Similarly, there was a difference in the p,p'-DDE exposure effect on Y-sperm proportion between the AHRR P185A and AHR R554K genotypes. The most pronounced effect of POP exposure was seen in subjects with <22 AR CAG repeats and the AHRR P185A G-allele. When categorizing the CB-153 and p,p'-DDE concentrations, we did not find any statistically significant interaction between exposure and genotype in relation to Y-sperm proportion, most likely because of the loss of power due to small numbers in each group. However, categorized data showed the same trend as uncategorized, namely a more pronounced increase in Y-sperm in men with short CAG repeats and the AHRR P185A G-allele.
Although we found a statistically significant interaction between the genotype and exposure levels in relation to the proportion of Y-sperm, the mechanisms behind these findings remain yet unresolved, since the biological processes regulating the Y : X chromosome ratio in sperm are yet unknown. For example, in the low p,p'-DDE exposure group, subjects carrying a short CAG repeat in combination with an AHRR G-allele had somewhat lower proportion of Y-sperm when compared with those with other AR and AHRR genotypes, whereas the situation was in the opposite direction looking at the men in the highest exposed p,p'-DDE group.
Current findings, which demonstrate modulating effects of androgen and dioxin-mediated pathways, are in agreement with epidemiological data relating offspring sex ratio or sperm Y : X ratio to exposure to POP biomarkers and also to biochemical data demonstrating that various POPs possess estrogenic, anti-estrogenic, dioxin-like and androgen competing properties (Kelce et al., 1995; Bonefeld Jørgensen et al., 1997, 2001; Danzo, 1997; Andersen et al., 2002). This endocrine-disrupting role has been argued to, at least partly, to account for the impairment of male reproductive function seen in several countries.
Although we found statistically significant interactions between AR and AHR/AHRR genotypes and POP exposure in relation to the Y : X chromosome ratio in sperm, the pattern of the trends does not allow us to draw any conclusions regarding the biological mechanisms involved. Albeit unknown, it is plausible that sex steroid mimicking actions and/or AHR binding affinity of various POPs may contribute to the changes in sperm Y : X ratio. On the basis of measurements of two POP biomarkers, CB-153 and p,p'-DDE, the results obtained in the current study support the surmise that both AR and AHR pathways may be involved in the bias in distribution of Y- and X-bearing spermatozoa following POP exposure. The major and persistent DDT metabolite p,p'-DDE is known to inhibit androgen binding to the androgen receptor and androgen-induced transcriptional activity (Kelce et al., 1995). Although this compound is not supposed to act through the AHR, due to a high correlation between serum concentration of different POPs (Jönsson et al., 2005) in the context of the present study, the levels of p,p'-DDE might be markers of exposure to other chemical compounds, binding to this receptor. For CB-153, no androgenic or anti-androgenic activity was shown by Bonefeld-Jørgensen et al. (2001). Instead, because of its non-planar structure, CB-153 has been demonstrated to antagonize the action of TCDD (Chen and Bunce, 2004).
Despite several association studies demonstrating the role of the polymorphic site R554K in susceptibility for health-related effects, the variant receptor has been shown to possess an equivalent ability to that of the wild-type receptor to bind to a dioxin-responsive element following treatment with TCDD and to stimulate CYP1A1 mRNA expression (Wong et al., 2001). Our data on R554K, showing a modifying effect in regard to the proportion of Y-sperm in ejaculate, are in agreement with the possibility that the variant allele at codon 554 could be associated with the alteration of important cellular processes other than CYP1A1 expression.
Regarding AHRR P185A, no functional studies have been performed and it is therefore unknown whether this SNP has a direct effect on AHR signalling in the testis or serves as a marker for some other functional polymorphism. Our findings are however in line with a recent study showing its association with the semen quality (Watanabe et al., 2004).
The results of this study seem to confirm our recent findings that the negative impact of POP exposure on sperm number and chromatin integrity is restricted to subjects with short CAG repeats (Giwercman et al., 2006). However, the design of this study does not permit any conclusions regarding the possible molecular mechanisms between the gene–environment interactions. It should be kept in mind that although we measured the levels of CB-153 and p,p'-DDE, these are to be considered as proxy markers of exposure to a broad range of POPs. CB-153 concentration was reported to be highly correlated with the total PCB concentration, the estimated TCDD equivalent (TEQ) from PCB, and the total POP-derived TEQ, respectively.
In summary, the major finding of this study is that genetic variants in the AR, AHR and AHRR genes affect spermatogenesis, expressed in terms of sperm Y : X ratio. To the best of our knowledge, this is the first study to indicate that the endocrine disrupting action of POPs, in relation to observed changes in sperm Y : X ratio, may be modulated by sex steroids and dioxin-mediated pathways. The outcome of the present study calls for consideration of the genetic background, which could influence the individual's susceptibility, when evaluating the adverse effects of these ubiquitous pollutants on male reproductive function.
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Acknowledgements |
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We wish to thank Camilla Anderberg and Kristina Lundin for excellent technical help and Kirsten Vang Nielsen from DakoCytomation for providing the PNA probes. This work was supported by grants from the European Commission (QLK4-CT-2001-00202), Swedish Research Council (grant no 521-2004-6072), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, Swedish government founding for clinical research, the Swedish Childhood Cancer Society (Grant No RKT04/001and 05/056) and Gunnar Nilsson Cancer Fund.
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References |
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