In Utero and Lactational Exposure to TCDD; Steroidogenic Outcomes Diff
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《毒物学科学杂志》
Department of Biology, Laboratory of Animal Physiology, 20014 University of Turku, Turku, Finland
Department of Physiology, University of Turku, 20520 Turku, Finland
Department of Environmental Health, Laboratory of Toxicology, National Public Health Institute, Box 95, 70701 Kuopio, Finland
Department of Pediatrics, University of Turku, 20520 Turku, Finland
Department of Anatomy, University of Turku, 20520 Turku, Finland
ABSTRACT
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has a potency to induce decreased fertility and structural reproductive anomalies in male and female mammals. While the activity profile of sex steroid hormone production distinctly differs in developing males and females, we wanted to analyze sex-specific effects of TCDD introduced in utero and via lactation on gonadal steroidogenesis and gonadotropin levels in male and female rat infant pups. One oral dose of TCDD (0, 0.04, 0.2, or 1.0 μg/kg) was given to dams on gestational day (GD) 13. Plasma testosterone, estradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and gonadal mRNA levels for steroid acute regulatory protein (StAR), cytochrome P-450 cholesterol side-chain cleavage (P450scc), 3-hydroxy-steroid-dehydrogenase/5-4 isomerase type I (3-HSD1), P-450 17-hydroxylase/17,20-lyase (P450–17), and cytochrome P-450 aromatase (P450arom) were determined on postnatal days (PND) 10–16. TCDD 1.0 μg/kg reduced body weights but did not affect relative testis weight or alter testicular and ovarian histology. Plasma estradiol levels in dams and female pups were reduced on PND 14 and 16. Progesterone levels remained unaltered, and FSH levels were increased in female pups. In males, testosterone levels were elevated on PND 10. Gonadal mRNA levels for StAR and steroidogenic enzymes increased during the postnatal growth. TCDD caused no changes in relatively low testicular mRNA levels. However, significant reductions in StAR and P450arom mRNA levels were seen in PND 14 ovaries, and P450arom activity was decreased in isolated ovarian follicles. We conclude that developing testis and male gonadotropin secretion are resistant to TCDD-induced toxicity. In female pups, reduced estradiol, ovarian P450arom expression and enzyme activity levels, and elevated FSH levels may have a role in the development of ovarian dysfunction reported in TCDD-exposed females.
Key Words: TCDD; steroidogenesis; follicle-stimulating hormone; FSH; luteinizing hormone; LH; mRNA; infant; ovary; testis; rat.
INTRODUCTION
Dioxins refer to a heterogeneous group of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic dioxin congener and the model compound of PCDD/Fs. TCDD has a wide range of toxic effects, including wasting syndrome, metabolic derailment, liver tumor promotion, and endocrine disruption causing reproductive and developmental defects. From the risk assessment point of view developmental defects of the reproductive system are among the most critical effects, because they have been observed at very low dose levels (Gray and Ostby, 1995; Mably et al., 1992a). The great majority of the toxic effects of TCDD are mediated through the aryl hydrocarbon receptor (AhR). Upon binding, the receptor–ligand complexes are translocated to the nucleus where they dimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT). By binding to the specific xenobiotic responsive transcriptional enhancer elements (XREs), the complex regulates the transcription of genes, e.g., for drug metabolizing enzymes. The aryl hardrocarbon receptor and ARNT have been localized in ovary (Khorram et al., 2002), testis (Schultz et al., 2003), and in brain areas regulating the secretion of gonadotropins (Huang et al., 2000). Acute high-dose effects of TCDD in adult rat gonads include testicular atrophy with decreased steroid hormone levels (Mebus et al., 1987; Rune et al., 1991). Anovulation and altered gonadotropin and estradiol (E2) levels are frequent in females (Chaffin et al., 1996; Gao et al., 2001).
Maternal exposure to TCDD may, in adult male and female progeny, result in genital dysmorphogenesis and impaired fertility (e.g., Flaws et al., 1997; Heimler et al., 1998; Salisbury and Marcinkiewicz, 2002). Because the elimination half-life of TCDD in female rats is 26 days (Li et al., 1995b), developing organs in animals exposed to TCDD in utero and via lactation are continuously exposed to TCDD. The developing reproductive tract is sensitive to TCDD, and the notion that the placental transfer of TCDD to offspring is much less effective compared to lactational transfer (Chen et al., 2001; Li et al., 1995b; Salisbury and Marcinkiewicz, 2002) emphasizes the exceptionally high dioxin sensitivity of the reproductive tract during fetal development.
Even though developmental exposure studies have confirmed adverse effects of TCDD on androgen-dependent growth of male accessory sex organs and epididymal sperm numbers (Gray et al., 1995, 1997a, 1997b; Mably et al., 1992a, 1992b; Simanainen et al., 2004), effects on testicular androgen synthesis have remained ambiguous (Cooke et al., 1998; Haavisto et al., 2001, in press; Roman et al., 1995). In female progeny, developmental reproductive toxicity of TCDD has been associated with deformed external genitalia and incomplete or delayed vaginal opening (Gray and Ostby, 1995), as well as with decreased ovulatory success (Li et al., 1995a; Petroff et al., 2003).
A number of cytochrome enzymes regulate steroid hormone production. These enzymes are potential targets of TCDD-induced toxicity. In vitro, TCDD has been shown to reduce the mRNA expression of cytochrome P-450 cholesterol side-chain cleavage (P450scc) and cytochrome P-450 aromatase (P450arom) enzymes in rat granulosa cells (Dasmahapatra et al., 2000), and the protein expression levels of cytochrome P-450 17-hydroxylase/17,20-lyase (P450–17) in human luteinized granulosa cells (Morán et al., 2003). Less, however, is known about in vivo effects of TCDD on the expression levels of steroidogenic enzymes.
In the present study, gonadal mRNA expression levels of steroid acute regulatory protein (StAR) and the selected key steroidogenic enzymes were analyzed in 10- to 16-day-old male and female rat infant pups. During this time period, prepubertal peak in serum FSH and estradiol (E2) levels occurs in female pups (Dhler et al., 1977). In developing male rats, the perinatal activity peak in steroidogenesis occurs prenatally (Habert and Picon, 1984), and the postnatal serum FSH, LH, and testosterone remain at relative low levels until puberty (El Gehani et al., 1998). However, because of observed stimulatory effects of TCDD in the prenatal rat testis (Haavisto et al., 2001), it is important to analyze whether the hormonal effect is visible in infant testis where fetal-type Leydig cells remain the prevailing steroidogenic cell population. In the present study, the exposure to non-fetolethal doses of TCDD was carried out on GD 13 when the first signs of sex-dependent morphological differentiation are detectable in the gonadal primordia but the gonads still are steroidogenically quiescent. As a result of different end-point effects of TCDD reported in male and female reproductive organs and reproductive physiology, the aim of the present study was to analyze whether sex-dependent differences can be recognized in infancy of in utero and lactationally exposed rat offspring.
MATERIALS AND METHODS
Animals.
Sprague-Dawley rats (Harlan, Zeist, The Netherlands) were housed individually in plastic cages with wire-mesh covers in a room with a 12-h light:dark cycle at 21±1°C and with 50 ± 10% relative humidity. Aspen-chips (Tapvei Co., Kaavi, Finland) were used as bedding and nesting material. Rats had free access to standard pelleted laboratory animal feed (R36, Ewos, Sdertelje, Sweden) and tap water. The Animal Experiment Committee of the University of Kuopio approved the experimental protocol.
Treatments.
Timed pregnant rats were randomly assigned to control and experimental groups (n 10 per group). The day when sperm was found in the vagina was considered as gestational day (GD) 0. TCDD (Ufa-Oil Institute, Russia), >99% pure as assessed by gas chromatography–mass spectrometry, was dissolved in diethyl ether, and adjusted volumes of the solution were mixed with corn oil after which the ether was allowed to evaporate. Diethyl ether and corn oil were of analytical grade and purchased from Merck (Darmstadt, Germany) and from BDH Laboratory Supplies (Poole, England), respectively. Before maternal dosing, the solution was carefully mixed in a magnetic stirrer and sonicated for 20 min. A single dose of TCDD (0, 0.04, 0.2, or 1.0 g/kg) in corn oil (4 ml/kg) was given by gavage on GD 13. Presumed exposure continued via lactation until the sample collection.
Sampling.
The day of birth was considered PND 0. One day after birth the litter size was adjusted to 4 male and 4 female pups to allow uniform lactational exposure. From controls and pups exposed to 1 μg/kg TCDD, samples were collected on PND 10, 12, 14, and 16. For dose–response analysis, samples from pups exposed to doses of 0.04 and 0.2 μg TCDD/kg were taken on PND 14. Dams and pups were anesthetized using a mixture of carbon dioxide and oxygen; they were then weighed and blood samples were collected in heparinized syringes. Blood kept on ice was centrifuged for 5 min at 1000 x g at +4°C. Plasma was stored at –20°C for the measurement of progesterone (P4), testosterone (T), E2, LH, and FSH levels. Ovaries and testes were excised, snap frozen in liquid nitrogen, and stored at –70°C.
For histology, 14-day-old testes and ovaries were fixed in 5% glutaraldehyde buffered in 0.16 mol/l S-collidine-HCl (pH 7.4) and postfixed with potassium ferrocyanide-osmium fixative. Dehydrated tissue pieces were embedded in Epon, and 1-μm-thick sections were stained with a 0.5% toluidine blue solution. Testis and ovarian histology and the number of developing ovarian follicles were evaluated under a light microscope from control and exposed (TCDD 1 μg/kg) pups. Gonads from five animals in each group were analyzed.
Hormone measurements.
T, P4, and E2 were measured from diethyl ether extracts of heparin plasma by time-resolved fluoroimmunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) as described elsewhere (Haavisto et al., 2001). Serum LH and FSH concentrations were determined by two-site time-resolved immunofluorometric assays (DELFIA) for rat LH and FSH. The assay detection limit was 0.100 ng/ml for T, 0.250 ng/ml for P4, 0.014 ng/ml for E2, 0.040 ng/ml for LH, and 0.100 ng/ml for FSH. The intra- and inter-assay variations were under 6% and 12%, respectively. For testosterone, the enhancement of assay sensitivity from 0.100 ng/ml to 0.040 ng/ml was obtained by an additional dilution of the commercial tracer and antisera to 5/8 from their original concentrations.
Ex vivo aromatase assay.
Dose–response analysis of TCDD-induced changes in ovarian P450arom enzyme activity was determined ex vivo in the isolated PND 14 ovarian follicles by measuring the incorporation of tritium from 1, 2-3H-androstenedione (NEN, Zaventem, Belgium) into water phase as described elsewhere (Lephart and Simpson, 1991). Ovarian follicles were isolated and cultured for 5 days in the presence of 1.1 IU of hFSH as described (Myllymki et al., 2005). After 4 days in culture, 10 pM of 1,2-3H-labeled androstenedione was added to culture medium. Radioactivity of ether-extracted water phase was measured using a Microbeta scintillation counter (Perkin Elmer Wallac).
Two-step real-time RT-PCR.
Total RNA was extracted from snap-frozen testes and ovaries with RNeasy Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. The tissue was homogenized with motor-driven plastic Eppendorf pestles, and DNA was subsequently sheared using Qia-shredder columns (Qiagen). Two micrograms of total RNA was reverse transcribed using Avian Myeloblastosis Virus (AMV, 30 U), Reverse Transcriptase Reaction Buffer (Promega), 1 μg Oligo(dT)15 Primer (Promega), and 40 units of RNase inhibitor (RNasin, Promega) in a final volume of 25 μl. For quantification of P450scc, P450–17, P450arom, 3-hydroxy-steroid-dehydrogenase/5-4 isomerase type I (3-HSD1), StAR, and S26 cDNA transcripts, real-time PCR was performed with specific primer pairs (Table 1). The annealing temperature was 57°C, and the reactions were performed using the QuantiTect SYRB-Green RT-PCR Kit (Qiagen) according to the manufacturer's instructions, which included use of the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with the use of continuous fluorescence detection. Ribosomal S26 was included as the endogenous normalization control for the amount of loaded RNA.
Statistical analysis of data.
All analyses were done with SPSS 11.0 software for Windows, and comparisons between treatment groups were carried out using one-way analysis of variance (ANOVA), followed by Dunnett's Pairwise Multiple Comparison t-test. Data were analyzed using litter mean values. A p value less than 0.05 was considered the limit for statistical significance.
RESULTS
Body and Testis Weight
TCDD had no effects on the litter size or the time of delivery; nor did TCDD treatment affect the body weight of dams (Table 2). In utero and lactational exposure to 1.0 μg/kg TCDD, however, significantly decreased the body weight of female offspring on PND 10, 12, 14, and 16. The decline from controls was 6.6%, 8.6%, 12.6%, and 12.0%, respectively. In male offspring, the decline in body weight was significant on PND12 (9.6%), PND 14 (9.2%), and PND 16 (21%). Dose–response analysis in 14-day-old offspring revealed growth retardation in male and female offspring only at the 1.0-μg/kg TCDD dose. A significant decrease (18.2 %) in the absolute testis weight was recorded at the dose of 1.0 μg/kg TCDD on PND 16. Relative testis weights at the age of 10 days were as in controls, but on PND 12 at 1.0 μg/kg TCDD and on PND 14 at 0.2 μg/kg they were moderately (6–8 %, p < 0.05) increased. Under histological evaluation, no structural differences were seen between the control PND 14 testes and those exposed to 1.0 μg/kg TCDD (Fig. 1 a, b). In PND 14 ovaries, structural changes were not observed, and no statistical differences were found in the number of primordial, primary, secondary, and small antral follicles (Fig. 1 c, d).
Plasma Estradiol, Progesterone, and Testosterone Levels
Plasma E2 levels measured from the dams on PND 14 and PND 16 were decreased, but the decline was significant (p < 0.05) only on PND 14 at the highest dose of TCDD (Table 3). P4 levels in the dams on day 14 were above the control level in each exposure group, but because of a relatively high inter-individual variation none of the changes were statistically significant. In female pups, plasma E2 levels were significantly decreased at 1 μg/kg on PND 14 (p < 0.05) and PND 16 (p < 0.01). Plasma P4 levels in the TCDD-exposed female pups were at the control level, and testosterone levels were under the detection limit throughout the study. Male testosterone levels were at the control level except for a sporadic increase (p < 0.05) observed in 10-day-old individuals exposed to 1.0 μg TCDD/kg.
Plasma FSH and LH levels
In dams, plasma LH and FSH levels were measured on PND 14 and PND 16 (Table 4). Only FSH showed a significant (p < 0.05) increase on PND 16 at 1.0 μg/kg. In female pups exposed to TCDD 1.0 μg/kg, LH was significantly (p < 0.05) increased on PND 14, and the values in 12- and 16-day-old females were above the control level, although the differences were not statistically significant. The FSH level was significantly elevated on PND 12 (p < 0.01) and PND 14 (p < 0.001). In males, relatively low plasma gonadotropin levels remained unaltered.
Steroidogenic Enzyme and StAR mRNA Levels
Except for P450arom, mRNA levels of steroidogenic enzymes quantified by RT-PCR increased in control animals between PND 10 and PND 14 (Fig. 2). Compared to developing ovaries, relative mRNA levels for P450scc, 3-HSD1, and P450-17 were lower in the corresponding testes, and none of those were affected by the exposure to TCDD (Fig. 2). Testicular P450arom mRNA levels were low, just above the detection limit. In ovaries, linear increase observed in P450scc, 3-HSD1, and P450-17 mRNA contents leveled off between PND 14 and PND 16. A dose of 1.0 μg/kg TCDD also caused a consistent, although statistically nonsignificant decrease in 3-HSD1 mRNA levels (Fig. 2). For ovarian P450scc mRNA, a significant (p < 0.05) decrease was seen in 16-day-old females exposed to 1.0 μg /kg TCDD. Dose–response analysis in 14-day-old females revealed a decrease in P450arom and StAR mRNA levels, and the change was significant in the group exposed to 0.2 μg TCDD/kg (Fig. 3A). No changes were seen in P450scc, P450–17, and 3-HSD1 mRNA levels.
Ex Vivo Aromatase Activity
In the isolated, maternally TCDD-exposed PND 14 ovarian follicles aromatase enzyme activity decreased dose-dependently (Fig. 3B). Compared to controls, the inhibition was statistically significant (p < 0.05) at 1.0 μg/kg dose of TCDD.
DISCUSSION
The results of the present study suggest that the growth and androgen production of infant rat testis are not particularly sensitive to non-fetolethal doses of TCDD, whereas such exposure has a greater potency to alter circulating gonadotropin and sex steroid hormone levels in female progeny. Females, in general, are known to exhibit higher sensitivity to TCDD (Beatty et al., 1978). This may be due to the longer half-life and higher accumulation of TCDD in female tissues, including ovaries (Li et al., 1995b). In addition, high levels of alpha fetoprotein in postnatal female rat circulation may potentiate toxicity of TCDD (Sotnichenko et al., 1999).
Reductions in the offspring body weight, a feature of TCDD toxicity (Birnbaum, 1995; Pohjanvirta and Tuomisto, 1994), confirmed the effectiveness of the present exposure regime. Maternally introduced TCDD (1–2 μg/kg) has also been shown to reduce absolute testis weight in adults (Gray et al., 1995, 1997a, 1997b; Mably et al., 1992b; Theobald et al., 2000; Wilker et al., 1996). Relative testis weights, however, may remain unaltered (Theobald et al., 2000; Wilker et al., 1996) or, as shown in the present study, may even be increased. This may suggest an initial compensatory response and/or reflect decreases in body weights. In the ovary, the absence of histological changes is in line with earlier reports showing unaltered numbers of primordial, primary, preantral, and small antral follicles in TCDD-exposed female pups (Salisbury and Marcinkiewicz, 2002). As a result of specific growth-retarding effects on large preovulatory follicles (Gray and Ostby, 1995; Salisbury and Marcinkiewicz, 2002) TCDD may, however, reduce the size and structure of mature ovaries.
The present findings in the male infants corroborate the results of Cooke et al. (1998), showing that developmental exposure to TCDD does not alter testosterone production in 2-week-old Sprague-Dawley male rats. In this study, resistance of developing testis to non-fetolethal doses of TCDD was further confirmed by unaltered gonadotropin levels and mRNA levels of StAR, 3-HSD1, P450scc, and P450-17. Studies in adult male rats have not found a significant impact of TCDD on the synthesis and secretion of gonadotropins (Bookstaff et al., 1990). Unexpectedly, the present study indicates that in 10-day-old pups and in prenatal rats (Haavisto et al., 2001), developmental exposure to TCDD may slightly increase testicular testosterone production. In adult male rats exposed to 50 μg TCDD/kg (Ruangwises et al., 1991) and in juvenile male rats exposed in utero and via lactation to 1 μg/kg TCDD (Roman et al., 1995), and in mouse Leydig tumor cells (Wilker et al., 1995), human chorionic gonadotropin (hCG)-stimulation has been shown to normalize TCDD-induced depression of testosterone levels. Therefore, the present and earlier studies in male rats (Haavisto et al., 2001; Simanainen et al., 2004) suggest that adverse effects reported in adult male sex accessory organs and epididymal sperm numbers may more likely be associated with changes in androgen receptor–mediated signaling pathways than with altered testosterone production in the developing testis. Undoubtedly, acute high-dose toxic effects of TCDD comprise testicular atrophy and altered levels of steroid-metabolizing enzymes, like P450scc (Moore et al., 1991).
The present results of the ovarian mRNA levels of StAR and steroidogenic enzymes correlate well with the reported postnatal steroidogenic profile characterized by high estradiol and FSH levels and relatively low LH levels in intact female offspring (Dhler and Wuttke, 1975; Herath et al., 2001). A significant rise in FSH levels between PND 10 and PND 20 is considered crucial for the induction of the growth of the primary and secondary follicle pools and cyclicity of female reproductive functions (Arendsen de Wolff-Exalto, 1982). A concomitant peak in plasma FSH and E2 levels in 14- to 15- day-old female rats (Herath et al., 2001) possibly results from nonfunctional inhibin regulation of FSH (Rivier and Vale, 1987) and a weakly developed estrogen-dependent negative feedback system (Kawagoe and Hiroi, 1983). The observed TCDD-induced increase in infant female FSH levels has a potential link to the attenuated and developmentally premature gonadotropin release and ovulation failures described in TCDD-exposed female rats (Gray and Ostby, 1995; Gao et al., 2001; Li et al., 1995a; Salisbury and Marcinkiewicz, 2002). In immature female rats, TCDD has been shown to directly affect the hypothalamo-pituitary axis by causing too early, i.e., premature, release of FSH and LH (Li et al., 1995a; Petroff et al., 2003). The mechanisms of TCDD-induced stimulation of gonadotropin release, however, have remained unresolved.
The observed decline in circulating estradiol levels in TCDD-exposed females is in line with the findings from other laboratories (Chaffin et al., 1996, 1997; Salisbury and Marcinkiewicz, 2002). The decrease in infant plasma estradiol levels in the presence of elevated FSH levels proposes ovarian-specific effects of TCDD. Stimulation of estradiol production in juvenile gonadotropin-primed female rats exposed to 10–60 μg/kg TCDD (Gao et al., 2001; Li et al., 1995a; Mizuyachi et al., 2002) suggests that the gonadotropin responsiveness of the developing ovary is relatively resistant to TCDD-induced changes.
The decrease in StAR mRNA, coding the protein needed to deliver cholesterol onto the inner mitochondrial membrane (Clark et al., 1994), was significant in 14-day-old females exposed to 0.2 μg TCDD/kg. The mechanism(s) of the putative downregulation of StAR transcripts by TCDD are not known. In the in vitro transfection assay, ligand-activated AhR has been shown to alter human StAR gene promoter activity (Sugawara et al., 2001).
A significant decrease in mRNA coding for P450scc, an enzyme catalyzing cholesterol side chain cleavage, was observed in 16-day-old females at 1.0 μg/kg TCDD. Like StAR (Mizutani et al., 1997; Ronen-Fuhrmann et al., 1998), P450scc is dominantly present in the infant rat theca cells and is upregulated in granulosa cells after gonadotropin stimulation (Ronen-Fuhrmann et al., 1998). The reported interference with P450scc activity in rat granulosa cells (Dasmahapatra et al., 2000) and in mouse (Fukuzawa et al., 2004) and rat testis (Kleeman et al., 1990) has raised cholesterol metabolism as a putative target of TCDD's steroidogenic action. However, in the present females the sporadic depression of P450scc mRNA levels apparently is not severe enough to interfere with pregnenolone formation. Progesterone levels remained at the control level, as did the mRNA levels of 3-HSD1. In cultured human lutenized granulosa cells, lyase activity has been considered one of the main targets of TCDD action (Morán et al., 2000). However, because if considerable variation in physiological status, direct mechanistic cause-and-effect comparisons of TCDD-induced effects in prepubertal, preovulatory, ovulatory, and post-ovulatory follicles or isolated follicle cells may not be feasible. In adult rat ovarian thecal cell and granulosa cell cultures, for instance, TCDD stimulates steroid hormone synthesis in thecal cells, whereas in granulosa-thecal cell co-cultures the effect is inhibitory (Grochowalski et al., 2001).
The cytochrome P-450 aromatase enzyme is the rate-limiting and FSH-dependent factor in estradiol synthesis. The results of the present study indicate that the levels of P450arom mRNA correlate well with low aromatase activity shown in the infant rat testes and with the relatively high peak enzyme activity values described also earlier in 10- to 16-day-old postnatal ovaries (George and Ojeda, 1987). In the TCDD-exposed 14-day-old female progeny, P450arom mRNA levels were significantly decreased at the 0.2 μg/kg dose, as was the enzyme activity at the 1.0 μg/kg dose of TCDD. Thus, aromatase gene expression seems to be a sensitive target of TCDD action. In cultured prepubertal and adult rat granulosa cells TCDD-induced elevation of CYP1A1 mRNA has been shown to parallel with a significant reduction in FSH-induced P450arom mRNA levels (Dasmahapatra et al., 2000). Binding sites for AhR have also been indicated in P450arom (cyp19) genes of various species (Tong et al., 2003). In human luteinizing granulosa cells, however, reduced estradiol production apparently is not due to altered aromatase enzyme production or enzyme activity (Heimler et al., 1998; Morán et al., 2000, 2003).
In summary, the present results propose that developmental exposure to TCDD exerts its most severe changes in infant female FSH secretion and ovarian estradiol synthesis, whereas testicular steroidogenesis and male gonadotropin secretion are relatively resistant to reproductive toxicity of TCDD. Further studies are needed to resolve the pituitary-gonadal effects of TCDD in developing female offspring. Among female steroidogenic enzymes, P450arom apparently is one of the most sensitive targets of TCDD-induced toxicity.
NOTES
2 These authors contributed equally to this study and should be considered as the first authors.
ACKNOWLEDGMENTS
We thank Ms. Arja Tamminen and Ms. Virpi Tiihonen for excellent technical assistance. This work was supported by the Finnish Cultural Foundation, Maj and Tor Nessling Foundation, the European Commission under the framework of the "Quality of Life" programme (Contracts: QLK4-CT1999–01422 ENVIR. REPROD.HEALTH, QLK4-2001–00269 EXPORED, and QLK4-CT2002–00603 EDEN), and the Academy of Finland (Grant number 77298).
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Department of Physiology, University of Turku, 20520 Turku, Finland
Department of Environmental Health, Laboratory of Toxicology, National Public Health Institute, Box 95, 70701 Kuopio, Finland
Department of Pediatrics, University of Turku, 20520 Turku, Finland
Department of Anatomy, University of Turku, 20520 Turku, Finland
ABSTRACT
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has a potency to induce decreased fertility and structural reproductive anomalies in male and female mammals. While the activity profile of sex steroid hormone production distinctly differs in developing males and females, we wanted to analyze sex-specific effects of TCDD introduced in utero and via lactation on gonadal steroidogenesis and gonadotropin levels in male and female rat infant pups. One oral dose of TCDD (0, 0.04, 0.2, or 1.0 μg/kg) was given to dams on gestational day (GD) 13. Plasma testosterone, estradiol, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH), and gonadal mRNA levels for steroid acute regulatory protein (StAR), cytochrome P-450 cholesterol side-chain cleavage (P450scc), 3-hydroxy-steroid-dehydrogenase/5-4 isomerase type I (3-HSD1), P-450 17-hydroxylase/17,20-lyase (P450–17), and cytochrome P-450 aromatase (P450arom) were determined on postnatal days (PND) 10–16. TCDD 1.0 μg/kg reduced body weights but did not affect relative testis weight or alter testicular and ovarian histology. Plasma estradiol levels in dams and female pups were reduced on PND 14 and 16. Progesterone levels remained unaltered, and FSH levels were increased in female pups. In males, testosterone levels were elevated on PND 10. Gonadal mRNA levels for StAR and steroidogenic enzymes increased during the postnatal growth. TCDD caused no changes in relatively low testicular mRNA levels. However, significant reductions in StAR and P450arom mRNA levels were seen in PND 14 ovaries, and P450arom activity was decreased in isolated ovarian follicles. We conclude that developing testis and male gonadotropin secretion are resistant to TCDD-induced toxicity. In female pups, reduced estradiol, ovarian P450arom expression and enzyme activity levels, and elevated FSH levels may have a role in the development of ovarian dysfunction reported in TCDD-exposed females.
Key Words: TCDD; steroidogenesis; follicle-stimulating hormone; FSH; luteinizing hormone; LH; mRNA; infant; ovary; testis; rat.
INTRODUCTION
Dioxins refer to a heterogeneous group of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic dioxin congener and the model compound of PCDD/Fs. TCDD has a wide range of toxic effects, including wasting syndrome, metabolic derailment, liver tumor promotion, and endocrine disruption causing reproductive and developmental defects. From the risk assessment point of view developmental defects of the reproductive system are among the most critical effects, because they have been observed at very low dose levels (Gray and Ostby, 1995; Mably et al., 1992a). The great majority of the toxic effects of TCDD are mediated through the aryl hydrocarbon receptor (AhR). Upon binding, the receptor–ligand complexes are translocated to the nucleus where they dimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT). By binding to the specific xenobiotic responsive transcriptional enhancer elements (XREs), the complex regulates the transcription of genes, e.g., for drug metabolizing enzymes. The aryl hardrocarbon receptor and ARNT have been localized in ovary (Khorram et al., 2002), testis (Schultz et al., 2003), and in brain areas regulating the secretion of gonadotropins (Huang et al., 2000). Acute high-dose effects of TCDD in adult rat gonads include testicular atrophy with decreased steroid hormone levels (Mebus et al., 1987; Rune et al., 1991). Anovulation and altered gonadotropin and estradiol (E2) levels are frequent in females (Chaffin et al., 1996; Gao et al., 2001).
Maternal exposure to TCDD may, in adult male and female progeny, result in genital dysmorphogenesis and impaired fertility (e.g., Flaws et al., 1997; Heimler et al., 1998; Salisbury and Marcinkiewicz, 2002). Because the elimination half-life of TCDD in female rats is 26 days (Li et al., 1995b), developing organs in animals exposed to TCDD in utero and via lactation are continuously exposed to TCDD. The developing reproductive tract is sensitive to TCDD, and the notion that the placental transfer of TCDD to offspring is much less effective compared to lactational transfer (Chen et al., 2001; Li et al., 1995b; Salisbury and Marcinkiewicz, 2002) emphasizes the exceptionally high dioxin sensitivity of the reproductive tract during fetal development.
Even though developmental exposure studies have confirmed adverse effects of TCDD on androgen-dependent growth of male accessory sex organs and epididymal sperm numbers (Gray et al., 1995, 1997a, 1997b; Mably et al., 1992a, 1992b; Simanainen et al., 2004), effects on testicular androgen synthesis have remained ambiguous (Cooke et al., 1998; Haavisto et al., 2001, in press; Roman et al., 1995). In female progeny, developmental reproductive toxicity of TCDD has been associated with deformed external genitalia and incomplete or delayed vaginal opening (Gray and Ostby, 1995), as well as with decreased ovulatory success (Li et al., 1995a; Petroff et al., 2003).
A number of cytochrome enzymes regulate steroid hormone production. These enzymes are potential targets of TCDD-induced toxicity. In vitro, TCDD has been shown to reduce the mRNA expression of cytochrome P-450 cholesterol side-chain cleavage (P450scc) and cytochrome P-450 aromatase (P450arom) enzymes in rat granulosa cells (Dasmahapatra et al., 2000), and the protein expression levels of cytochrome P-450 17-hydroxylase/17,20-lyase (P450–17) in human luteinized granulosa cells (Morán et al., 2003). Less, however, is known about in vivo effects of TCDD on the expression levels of steroidogenic enzymes.
In the present study, gonadal mRNA expression levels of steroid acute regulatory protein (StAR) and the selected key steroidogenic enzymes were analyzed in 10- to 16-day-old male and female rat infant pups. During this time period, prepubertal peak in serum FSH and estradiol (E2) levels occurs in female pups (Dhler et al., 1977). In developing male rats, the perinatal activity peak in steroidogenesis occurs prenatally (Habert and Picon, 1984), and the postnatal serum FSH, LH, and testosterone remain at relative low levels until puberty (El Gehani et al., 1998). However, because of observed stimulatory effects of TCDD in the prenatal rat testis (Haavisto et al., 2001), it is important to analyze whether the hormonal effect is visible in infant testis where fetal-type Leydig cells remain the prevailing steroidogenic cell population. In the present study, the exposure to non-fetolethal doses of TCDD was carried out on GD 13 when the first signs of sex-dependent morphological differentiation are detectable in the gonadal primordia but the gonads still are steroidogenically quiescent. As a result of different end-point effects of TCDD reported in male and female reproductive organs and reproductive physiology, the aim of the present study was to analyze whether sex-dependent differences can be recognized in infancy of in utero and lactationally exposed rat offspring.
MATERIALS AND METHODS
Animals.
Sprague-Dawley rats (Harlan, Zeist, The Netherlands) were housed individually in plastic cages with wire-mesh covers in a room with a 12-h light:dark cycle at 21±1°C and with 50 ± 10% relative humidity. Aspen-chips (Tapvei Co., Kaavi, Finland) were used as bedding and nesting material. Rats had free access to standard pelleted laboratory animal feed (R36, Ewos, Sdertelje, Sweden) and tap water. The Animal Experiment Committee of the University of Kuopio approved the experimental protocol.
Treatments.
Timed pregnant rats were randomly assigned to control and experimental groups (n 10 per group). The day when sperm was found in the vagina was considered as gestational day (GD) 0. TCDD (Ufa-Oil Institute, Russia), >99% pure as assessed by gas chromatography–mass spectrometry, was dissolved in diethyl ether, and adjusted volumes of the solution were mixed with corn oil after which the ether was allowed to evaporate. Diethyl ether and corn oil were of analytical grade and purchased from Merck (Darmstadt, Germany) and from BDH Laboratory Supplies (Poole, England), respectively. Before maternal dosing, the solution was carefully mixed in a magnetic stirrer and sonicated for 20 min. A single dose of TCDD (0, 0.04, 0.2, or 1.0 g/kg) in corn oil (4 ml/kg) was given by gavage on GD 13. Presumed exposure continued via lactation until the sample collection.
Sampling.
The day of birth was considered PND 0. One day after birth the litter size was adjusted to 4 male and 4 female pups to allow uniform lactational exposure. From controls and pups exposed to 1 μg/kg TCDD, samples were collected on PND 10, 12, 14, and 16. For dose–response analysis, samples from pups exposed to doses of 0.04 and 0.2 μg TCDD/kg were taken on PND 14. Dams and pups were anesthetized using a mixture of carbon dioxide and oxygen; they were then weighed and blood samples were collected in heparinized syringes. Blood kept on ice was centrifuged for 5 min at 1000 x g at +4°C. Plasma was stored at –20°C for the measurement of progesterone (P4), testosterone (T), E2, LH, and FSH levels. Ovaries and testes were excised, snap frozen in liquid nitrogen, and stored at –70°C.
For histology, 14-day-old testes and ovaries were fixed in 5% glutaraldehyde buffered in 0.16 mol/l S-collidine-HCl (pH 7.4) and postfixed with potassium ferrocyanide-osmium fixative. Dehydrated tissue pieces were embedded in Epon, and 1-μm-thick sections were stained with a 0.5% toluidine blue solution. Testis and ovarian histology and the number of developing ovarian follicles were evaluated under a light microscope from control and exposed (TCDD 1 μg/kg) pups. Gonads from five animals in each group were analyzed.
Hormone measurements.
T, P4, and E2 were measured from diethyl ether extracts of heparin plasma by time-resolved fluoroimmunoassay (DELFIA, PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) as described elsewhere (Haavisto et al., 2001). Serum LH and FSH concentrations were determined by two-site time-resolved immunofluorometric assays (DELFIA) for rat LH and FSH. The assay detection limit was 0.100 ng/ml for T, 0.250 ng/ml for P4, 0.014 ng/ml for E2, 0.040 ng/ml for LH, and 0.100 ng/ml for FSH. The intra- and inter-assay variations were under 6% and 12%, respectively. For testosterone, the enhancement of assay sensitivity from 0.100 ng/ml to 0.040 ng/ml was obtained by an additional dilution of the commercial tracer and antisera to 5/8 from their original concentrations.
Ex vivo aromatase assay.
Dose–response analysis of TCDD-induced changes in ovarian P450arom enzyme activity was determined ex vivo in the isolated PND 14 ovarian follicles by measuring the incorporation of tritium from 1, 2-3H-androstenedione (NEN, Zaventem, Belgium) into water phase as described elsewhere (Lephart and Simpson, 1991). Ovarian follicles were isolated and cultured for 5 days in the presence of 1.1 IU of hFSH as described (Myllymki et al., 2005). After 4 days in culture, 10 pM of 1,2-3H-labeled androstenedione was added to culture medium. Radioactivity of ether-extracted water phase was measured using a Microbeta scintillation counter (Perkin Elmer Wallac).
Two-step real-time RT-PCR.
Total RNA was extracted from snap-frozen testes and ovaries with RNeasy Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. The tissue was homogenized with motor-driven plastic Eppendorf pestles, and DNA was subsequently sheared using Qia-shredder columns (Qiagen). Two micrograms of total RNA was reverse transcribed using Avian Myeloblastosis Virus (AMV, 30 U), Reverse Transcriptase Reaction Buffer (Promega), 1 μg Oligo(dT)15 Primer (Promega), and 40 units of RNase inhibitor (RNasin, Promega) in a final volume of 25 μl. For quantification of P450scc, P450–17, P450arom, 3-hydroxy-steroid-dehydrogenase/5-4 isomerase type I (3-HSD1), StAR, and S26 cDNA transcripts, real-time PCR was performed with specific primer pairs (Table 1). The annealing temperature was 57°C, and the reactions were performed using the QuantiTect SYRB-Green RT-PCR Kit (Qiagen) according to the manufacturer's instructions, which included use of the DNA Engine Opticon system (MJ Research, Inc., Waltham, MA) with the use of continuous fluorescence detection. Ribosomal S26 was included as the endogenous normalization control for the amount of loaded RNA.
Statistical analysis of data.
All analyses were done with SPSS 11.0 software for Windows, and comparisons between treatment groups were carried out using one-way analysis of variance (ANOVA), followed by Dunnett's Pairwise Multiple Comparison t-test. Data were analyzed using litter mean values. A p value less than 0.05 was considered the limit for statistical significance.
RESULTS
Body and Testis Weight
TCDD had no effects on the litter size or the time of delivery; nor did TCDD treatment affect the body weight of dams (Table 2). In utero and lactational exposure to 1.0 μg/kg TCDD, however, significantly decreased the body weight of female offspring on PND 10, 12, 14, and 16. The decline from controls was 6.6%, 8.6%, 12.6%, and 12.0%, respectively. In male offspring, the decline in body weight was significant on PND12 (9.6%), PND 14 (9.2%), and PND 16 (21%). Dose–response analysis in 14-day-old offspring revealed growth retardation in male and female offspring only at the 1.0-μg/kg TCDD dose. A significant decrease (18.2 %) in the absolute testis weight was recorded at the dose of 1.0 μg/kg TCDD on PND 16. Relative testis weights at the age of 10 days were as in controls, but on PND 12 at 1.0 μg/kg TCDD and on PND 14 at 0.2 μg/kg they were moderately (6–8 %, p < 0.05) increased. Under histological evaluation, no structural differences were seen between the control PND 14 testes and those exposed to 1.0 μg/kg TCDD (Fig. 1 a, b). In PND 14 ovaries, structural changes were not observed, and no statistical differences were found in the number of primordial, primary, secondary, and small antral follicles (Fig. 1 c, d).
Plasma Estradiol, Progesterone, and Testosterone Levels
Plasma E2 levels measured from the dams on PND 14 and PND 16 were decreased, but the decline was significant (p < 0.05) only on PND 14 at the highest dose of TCDD (Table 3). P4 levels in the dams on day 14 were above the control level in each exposure group, but because of a relatively high inter-individual variation none of the changes were statistically significant. In female pups, plasma E2 levels were significantly decreased at 1 μg/kg on PND 14 (p < 0.05) and PND 16 (p < 0.01). Plasma P4 levels in the TCDD-exposed female pups were at the control level, and testosterone levels were under the detection limit throughout the study. Male testosterone levels were at the control level except for a sporadic increase (p < 0.05) observed in 10-day-old individuals exposed to 1.0 μg TCDD/kg.
Plasma FSH and LH levels
In dams, plasma LH and FSH levels were measured on PND 14 and PND 16 (Table 4). Only FSH showed a significant (p < 0.05) increase on PND 16 at 1.0 μg/kg. In female pups exposed to TCDD 1.0 μg/kg, LH was significantly (p < 0.05) increased on PND 14, and the values in 12- and 16-day-old females were above the control level, although the differences were not statistically significant. The FSH level was significantly elevated on PND 12 (p < 0.01) and PND 14 (p < 0.001). In males, relatively low plasma gonadotropin levels remained unaltered.
Steroidogenic Enzyme and StAR mRNA Levels
Except for P450arom, mRNA levels of steroidogenic enzymes quantified by RT-PCR increased in control animals between PND 10 and PND 14 (Fig. 2). Compared to developing ovaries, relative mRNA levels for P450scc, 3-HSD1, and P450-17 were lower in the corresponding testes, and none of those were affected by the exposure to TCDD (Fig. 2). Testicular P450arom mRNA levels were low, just above the detection limit. In ovaries, linear increase observed in P450scc, 3-HSD1, and P450-17 mRNA contents leveled off between PND 14 and PND 16. A dose of 1.0 μg/kg TCDD also caused a consistent, although statistically nonsignificant decrease in 3-HSD1 mRNA levels (Fig. 2). For ovarian P450scc mRNA, a significant (p < 0.05) decrease was seen in 16-day-old females exposed to 1.0 μg /kg TCDD. Dose–response analysis in 14-day-old females revealed a decrease in P450arom and StAR mRNA levels, and the change was significant in the group exposed to 0.2 μg TCDD/kg (Fig. 3A). No changes were seen in P450scc, P450–17, and 3-HSD1 mRNA levels.
Ex Vivo Aromatase Activity
In the isolated, maternally TCDD-exposed PND 14 ovarian follicles aromatase enzyme activity decreased dose-dependently (Fig. 3B). Compared to controls, the inhibition was statistically significant (p < 0.05) at 1.0 μg/kg dose of TCDD.
DISCUSSION
The results of the present study suggest that the growth and androgen production of infant rat testis are not particularly sensitive to non-fetolethal doses of TCDD, whereas such exposure has a greater potency to alter circulating gonadotropin and sex steroid hormone levels in female progeny. Females, in general, are known to exhibit higher sensitivity to TCDD (Beatty et al., 1978). This may be due to the longer half-life and higher accumulation of TCDD in female tissues, including ovaries (Li et al., 1995b). In addition, high levels of alpha fetoprotein in postnatal female rat circulation may potentiate toxicity of TCDD (Sotnichenko et al., 1999).
Reductions in the offspring body weight, a feature of TCDD toxicity (Birnbaum, 1995; Pohjanvirta and Tuomisto, 1994), confirmed the effectiveness of the present exposure regime. Maternally introduced TCDD (1–2 μg/kg) has also been shown to reduce absolute testis weight in adults (Gray et al., 1995, 1997a, 1997b; Mably et al., 1992b; Theobald et al., 2000; Wilker et al., 1996). Relative testis weights, however, may remain unaltered (Theobald et al., 2000; Wilker et al., 1996) or, as shown in the present study, may even be increased. This may suggest an initial compensatory response and/or reflect decreases in body weights. In the ovary, the absence of histological changes is in line with earlier reports showing unaltered numbers of primordial, primary, preantral, and small antral follicles in TCDD-exposed female pups (Salisbury and Marcinkiewicz, 2002). As a result of specific growth-retarding effects on large preovulatory follicles (Gray and Ostby, 1995; Salisbury and Marcinkiewicz, 2002) TCDD may, however, reduce the size and structure of mature ovaries.
The present findings in the male infants corroborate the results of Cooke et al. (1998), showing that developmental exposure to TCDD does not alter testosterone production in 2-week-old Sprague-Dawley male rats. In this study, resistance of developing testis to non-fetolethal doses of TCDD was further confirmed by unaltered gonadotropin levels and mRNA levels of StAR, 3-HSD1, P450scc, and P450-17. Studies in adult male rats have not found a significant impact of TCDD on the synthesis and secretion of gonadotropins (Bookstaff et al., 1990). Unexpectedly, the present study indicates that in 10-day-old pups and in prenatal rats (Haavisto et al., 2001), developmental exposure to TCDD may slightly increase testicular testosterone production. In adult male rats exposed to 50 μg TCDD/kg (Ruangwises et al., 1991) and in juvenile male rats exposed in utero and via lactation to 1 μg/kg TCDD (Roman et al., 1995), and in mouse Leydig tumor cells (Wilker et al., 1995), human chorionic gonadotropin (hCG)-stimulation has been shown to normalize TCDD-induced depression of testosterone levels. Therefore, the present and earlier studies in male rats (Haavisto et al., 2001; Simanainen et al., 2004) suggest that adverse effects reported in adult male sex accessory organs and epididymal sperm numbers may more likely be associated with changes in androgen receptor–mediated signaling pathways than with altered testosterone production in the developing testis. Undoubtedly, acute high-dose toxic effects of TCDD comprise testicular atrophy and altered levels of steroid-metabolizing enzymes, like P450scc (Moore et al., 1991).
The present results of the ovarian mRNA levels of StAR and steroidogenic enzymes correlate well with the reported postnatal steroidogenic profile characterized by high estradiol and FSH levels and relatively low LH levels in intact female offspring (Dhler and Wuttke, 1975; Herath et al., 2001). A significant rise in FSH levels between PND 10 and PND 20 is considered crucial for the induction of the growth of the primary and secondary follicle pools and cyclicity of female reproductive functions (Arendsen de Wolff-Exalto, 1982). A concomitant peak in plasma FSH and E2 levels in 14- to 15- day-old female rats (Herath et al., 2001) possibly results from nonfunctional inhibin regulation of FSH (Rivier and Vale, 1987) and a weakly developed estrogen-dependent negative feedback system (Kawagoe and Hiroi, 1983). The observed TCDD-induced increase in infant female FSH levels has a potential link to the attenuated and developmentally premature gonadotropin release and ovulation failures described in TCDD-exposed female rats (Gray and Ostby, 1995; Gao et al., 2001; Li et al., 1995a; Salisbury and Marcinkiewicz, 2002). In immature female rats, TCDD has been shown to directly affect the hypothalamo-pituitary axis by causing too early, i.e., premature, release of FSH and LH (Li et al., 1995a; Petroff et al., 2003). The mechanisms of TCDD-induced stimulation of gonadotropin release, however, have remained unresolved.
The observed decline in circulating estradiol levels in TCDD-exposed females is in line with the findings from other laboratories (Chaffin et al., 1996, 1997; Salisbury and Marcinkiewicz, 2002). The decrease in infant plasma estradiol levels in the presence of elevated FSH levels proposes ovarian-specific effects of TCDD. Stimulation of estradiol production in juvenile gonadotropin-primed female rats exposed to 10–60 μg/kg TCDD (Gao et al., 2001; Li et al., 1995a; Mizuyachi et al., 2002) suggests that the gonadotropin responsiveness of the developing ovary is relatively resistant to TCDD-induced changes.
The decrease in StAR mRNA, coding the protein needed to deliver cholesterol onto the inner mitochondrial membrane (Clark et al., 1994), was significant in 14-day-old females exposed to 0.2 μg TCDD/kg. The mechanism(s) of the putative downregulation of StAR transcripts by TCDD are not known. In the in vitro transfection assay, ligand-activated AhR has been shown to alter human StAR gene promoter activity (Sugawara et al., 2001).
A significant decrease in mRNA coding for P450scc, an enzyme catalyzing cholesterol side chain cleavage, was observed in 16-day-old females at 1.0 μg/kg TCDD. Like StAR (Mizutani et al., 1997; Ronen-Fuhrmann et al., 1998), P450scc is dominantly present in the infant rat theca cells and is upregulated in granulosa cells after gonadotropin stimulation (Ronen-Fuhrmann et al., 1998). The reported interference with P450scc activity in rat granulosa cells (Dasmahapatra et al., 2000) and in mouse (Fukuzawa et al., 2004) and rat testis (Kleeman et al., 1990) has raised cholesterol metabolism as a putative target of TCDD's steroidogenic action. However, in the present females the sporadic depression of P450scc mRNA levels apparently is not severe enough to interfere with pregnenolone formation. Progesterone levels remained at the control level, as did the mRNA levels of 3-HSD1. In cultured human lutenized granulosa cells, lyase activity has been considered one of the main targets of TCDD action (Morán et al., 2000). However, because if considerable variation in physiological status, direct mechanistic cause-and-effect comparisons of TCDD-induced effects in prepubertal, preovulatory, ovulatory, and post-ovulatory follicles or isolated follicle cells may not be feasible. In adult rat ovarian thecal cell and granulosa cell cultures, for instance, TCDD stimulates steroid hormone synthesis in thecal cells, whereas in granulosa-thecal cell co-cultures the effect is inhibitory (Grochowalski et al., 2001).
The cytochrome P-450 aromatase enzyme is the rate-limiting and FSH-dependent factor in estradiol synthesis. The results of the present study indicate that the levels of P450arom mRNA correlate well with low aromatase activity shown in the infant rat testes and with the relatively high peak enzyme activity values described also earlier in 10- to 16-day-old postnatal ovaries (George and Ojeda, 1987). In the TCDD-exposed 14-day-old female progeny, P450arom mRNA levels were significantly decreased at the 0.2 μg/kg dose, as was the enzyme activity at the 1.0 μg/kg dose of TCDD. Thus, aromatase gene expression seems to be a sensitive target of TCDD action. In cultured prepubertal and adult rat granulosa cells TCDD-induced elevation of CYP1A1 mRNA has been shown to parallel with a significant reduction in FSH-induced P450arom mRNA levels (Dasmahapatra et al., 2000). Binding sites for AhR have also been indicated in P450arom (cyp19) genes of various species (Tong et al., 2003). In human luteinizing granulosa cells, however, reduced estradiol production apparently is not due to altered aromatase enzyme production or enzyme activity (Heimler et al., 1998; Morán et al., 2000, 2003).
In summary, the present results propose that developmental exposure to TCDD exerts its most severe changes in infant female FSH secretion and ovarian estradiol synthesis, whereas testicular steroidogenesis and male gonadotropin secretion are relatively resistant to reproductive toxicity of TCDD. Further studies are needed to resolve the pituitary-gonadal effects of TCDD in developing female offspring. Among female steroidogenic enzymes, P450arom apparently is one of the most sensitive targets of TCDD-induced toxicity.
NOTES
2 These authors contributed equally to this study and should be considered as the first authors.
ACKNOWLEDGMENTS
We thank Ms. Arja Tamminen and Ms. Virpi Tiihonen for excellent technical assistance. This work was supported by the Finnish Cultural Foundation, Maj and Tor Nessling Foundation, the European Commission under the framework of the "Quality of Life" programme (Contracts: QLK4-CT1999–01422 ENVIR. REPROD.HEALTH, QLK4-2001–00269 EXPORED, and QLK4-CT2002–00603 EDEN), and the Academy of Finland (Grant number 77298).
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