- Email: [email protected]
Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat follicular steroidogenesis
Reproductive Toxicology 22 (2006) 521–528
Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat follicular steroidogenesis Sari A. Pesonen a,b,∗ , Tapio E. Haavisto a , Matti Viluksela c , Jorma Toppari b,d , Jorma Paranko e a
Department of Biology, Laboratory of Animal Physiology, University of Turku, 20014 Turku, Finland b Department of Physiology, University of Turku, 20520 Turku, Finland c Department of Environmental Health, Laboratory of Toxicology, National Public Health Institute, Box 95, 70701 Kuopio, Finland d Department of Pediatrics, University of Turku, 20520 Turku, Finland e Department of Anatomy, University of Turku, 20520 Turku, Finland Received 19 December 2005; received in revised form 8 February 2006; accepted 3 March 2006 Available online 18 May 2006
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant and causes adverse effects on female reproduction when administered to rats. Our aims were to study effects of gestational and lactational exposure to TCDD on ovarian steroidogenesis and steroidogenic enzyme expression of offspring on postnatal day (PND) 14 in the rat and sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro. Synthetic estrogen diethylstilbestrol (DES) was used as a treatment control. Serum progesterone (P4) level in offspring increased significantly on PND 14 in the TCDD (1 g/kg)-exposed group while body weight, FSH and E2 levels were not changed. In ovarian follicles of offspring on PND 14 in the TCDD-exposed groups, protein expression of cytochrome P-450 aromatase, cytochrome P-450 cholesterol side-chain cleavage, steroidogenic acute regulatory protein, 3-hydroxy-steroid-dehydrogenase/5 -4 isomerase type 1, or P4 receptor was not affected. TCDD decreased E2 and P4 production in ex vivo follicle culture. DES at a dose level of 0.1 mg/kg was dystocic while a dose 0.02 mg/kg increased ovarian ex vivo E2 and testosterone production without affecting P450arom activity indicating stimulation of early steps of steroidogenic pathway. Data suggests that TCDD has multiple targets in ovarian steroidogenesis, but the inhibitory action represented as decreased follicular steroid hormone production ex vivo is not apparent at the ovarian protein expression. Furthermore, TCDD had no direct effect on immature rat ovarian steroidogenesis in vitro suggesting that the follicle culture method is not a sensitive method to study the mechanisms of TCDD action. © 2006 Elsevier Inc. All rights reserved. Keywords: TCDD; DES; Ovary; Follicle; Steroidogenesis; Aromatase; Maternal exposure; Offspring
1. Introduction 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and highly toxic environmental pollutant and it bioaccumulates in the body due to its lipophilic properties and slow metabolism and excretion. There is an increasing evidence of TCDD’s endocrine disrupting effects on immature female reproductive system. In the hypothalamus, TCDD has been shown to decrease the responsiveness to estradiol [1]. A direct stimulatory effect of
∗
Corresponding author at: Department of Physiology, University of Turku, 20520 Turku, Finland. Tel.: +358 50 5730 433; fax: +358 2 3337352. E-mail address: [email protected] (S.A. Pesonen). 0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2006.03.007
TCDD on pituitary gonadotropin secretion in immature female rats was demonstrated by Li et al. [2]. In utero and lactational exposure to TCDD has been shown to disrupt estrus cycles and inhibit ovulation rate [3]. Furthermore, maternal exposure to TCDD has potency to directly disturb ovarian steroidogenesis of female progeny [4] and TCDD has been shown to alter the activity and expression of several ovarian steroidogenic enzymes either directly or indirectly [5–7]. Reproductive organs and endocrine system are sensitive to TCDD during development and sexual maturation [8,9]. Compared to placental transfer, exposure via lactation plays a major role in offspring receiving maternal PCDD/Fs and dioxin-like PCBs [10]. Female Sprague–Dawley rats appear to be more sensitive to dioxin-like PCBs than males [11]. It has been proposed,
522
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
that the increased susceptibility of females to TCDD toxicity may be due to the sex-specific kinetics of TCDD [12]. The sensitivity to TCDD has been demonstrated to vary with the alterations in ovarian hormonal status [13], and estrogens have been shown to modulate the effects exerted by TCDD [12,14]. Therefore, the intensive hormone metabolism in the ovary during sexual maturation at the age of 2 weeks may increase the susceptibility of infantile female rat to TCDD. Maternally introduced diethylstilbestrol (DES), was used as a treatment control. Maternal DES exposure has been shown to promote the reproductive functions of immature female rat by increasing plasma FSH concentration and by accelerating the development of primary and secondary follicles in the ovary [15]. In cultured ovarian cells, stimulatory effect of DES has been demonstrated by increased 17-HSD-activity, an enzyme converting androstenedione to testosterone and estrone to estradiol [16]. Also developmental toxicity of DES in terms of dystocia and decreased pup body weight has been reported [17,18]. The mechanism(s) of TCDD-induced alterations in the ovary are still unclear. Furthermore, little is known about low-dose effects of TCDD on immature ovarian steroidogenesis. We have previously demonstrated that in immature rat ovary, maternal TCDD decreases cytochrome P-450 aromatase enzyme (P450arom) activity, and mRNA levels of P450arom, cytochrome P-450 cholesterol side-chain cleavage (P450scc), and steroidogenic acute regulatory protein (StAR) [7]. In the present study, we studied effects of in utero and lactational exposure to TCDD on ovarian steroidogenesis ex vivo and protein expression of ovarian steroidogenic enzymes in offspring on postnatal day (PND) 14 in the rat. Furthermore, the sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro was studied. 2. Materials ad methods 2.1. Animals Timed mated Sprague–Dawley rats (Harlan, Zeist, The Netherlands) 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 0. Rats were housed individually in plastic cages with wire-mesh covers in a room with a 12-h light/12-h 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 tap water and standard pelleted laboratory animal feed R36 (Ewos, S¨odertelje, Sweden) in TCDD-exposures and Commercial RM1 (E) SQC (Special Diet Services, Witham, England) in DES-exposures, respectively. The day of birth was considered postnatal day (PND) 0. One day after birth the litter size was adjusted to eight pups by random culling to allow uniform lactational exposure. Dams were allowed to nurse offspring until the termination of the experiment on PND 14. The Animal Experiment Committee of the University of Kuopio and the Kuopio Provincial Government, and The Turku University Committee on Ethics of Animal Experimentation approved the experimental protocols.
2.2. TCDD treatments TCDD (Ufa-Oil Institute, Ufa, 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 let to evaporate. Dosing solutions were carefully mixed in a magnetic stirrer and sonicated for 20 min before dosing. Diethyl ether and corn oil were of analyt-
ical grade and purchased from Merck (Darmstadt, Germany) and from BDH Laboratory Supplies (Poole, England), respectively. A single maternal dose of TCDD (0.04, 0.2 or 1.0 g/kg) was given in 4 ml/kg corn oil by gavage on gestational day 13. Controls were given corn oil alone. Dose levels and the timing of exposure were similar to our previous parallel study showing that identical TCDD-exposure inhibited immature rat ovarian P450arom activity and mRNA levels of P450arom, P450scc, and StAR protein [7]. In the present study, we wanted to further study TCDD-induced ovarian steroidogenic inhibition.
2.3. DES treatments DES (Sigma, St. Louis, USA) was dissolved in dimethylsulphoxide (DMSO; Sigma) and adjusted volumes of the solutions were mixed with corn oil. Total of three subcutaneous injections of DES (0.004, 0.02 or 0.1 mg/kg) were given in the volume of 300 l corn oil on gestational days 13, 15 and 17. Controls were given DMSO in corn oil alone. In our earlier study, identical DES-exposure at a dose of 0.1 mg/kg decreased significantly testicular and plasma T content of male rat fetuses [19]. In this study, the highest dose level and the days for treatment were selected to study whether DES-induced steroidogenic disruption could be seen also in female gonads.
2.4. Sampling On the PND 14, individual body weight was recorded and pups were euthanized by cervical dislocation after mixture of carbon dioxide and oxygen anesthesia. Blood was obtained by cardiac puncture into heparinized syringes, kept on ice, and centrifuged for 5 min at 1000 × g at +4 ◦ C. Plasma was stored at −20 ◦ C before measurement of progesterone (P4), estradiol (E2), LH and FSH. Ovaries for Western blot were excised, snap frozen in liquid nitrogen, and stored at −70 ◦ C.
2.5. Follicle culture On the PND 14, ovaries were aseptically collected from offspring euthanized (see above) and given into sterile phosphate-buffered saline (PBS, pH 7.4) solution at RT. Follicles were isolated using the method of Cain et al. [20] with minor modifications described earlier [21]. Follicles, 15–50 for each tissue culture dish (Nunc, Roskilde, Denmark; 1.9 cm2 ) at final volume of 250 l were cultured in medium (McCoy’s 5A, without phenol red and sodium; Sigma) supplemented with 600 mg glucose, 1 ml penicillin–streptomycin, 200 l insulin, 500 g transferrin and 4 g hydrocortisone for each 100 ml of medium under humidified air containing 5% CO2 at 37 ◦ C. Human recombinant follicle-stimulating hormone (hFSH, courtesy of N.V. Organon, The Netherlands) was added to the well at the final concentration of 1.1 IU/250 l. On day 3, a sample of 100 l was taken from the medium and replaced with an equal volume of fresh medium. On day 5 at the end of the experiment, medium was collected and frozen at −20 ◦ C for analysis of E2, P4, and T. For TCDD (0, 0.01, 0.1, 1, 10 or 50 nM) and DES (100 ng/ml = 0.37 M) in vitro studies, the chemical was dissolved in DMSO (Sigma) and added to the culture medium in the beginning of the culture. The final concentration of DMSO in the culture medium was 1 l/ml both in DEStreated and control follicles. The viability of follicular cells was assayed at the end of the culture on the basis of tissue organization and with trypan-blue exclusion method as described earlier [21].
2.6. Aromatase assay P450arom activity was assayed by measuring the incorporation of tritium from [1,2-3 H]androstenedione (NEN, Zaventem, Belgium) into the water phase as described [22]. The in vitro effect of TCDD on ovarian P450arom activity was measured on PND 14 in follicles derived from female offspring whose mothers were not treated. Isolated follicles were cultured for 5 days in the presence of 1.1 IU of hFSH and TCDD at dose 0, 0.01, 0.1, 1, 10 or 50 nM. To study the effect of maternal DES exposure on ovarian P450arom activity of female pups, follicles isolated from control and DES-exposed (0.02 mg/kg) animals were cultured for 5 days in the presence of 1.1 IU of hFSH. On day 4, 10 pM of [1,2-3 H]-labelled androstenedione was added in both studies. After ether extraction of the medium, radioactivity of the water phase was measured
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528 using a Microbeta scintillation counter (Perkin-Elmer Wallac, Turku, Finland). For previously measured P450arom activity of ovarian follicles after maternal TCDD-exposure and in vitro DES treatment, see Section 3.
2.7. cAMP assay To measure the effects of gestational and lactational exposure to TCDD (0.04, 0.2 or 1.0 g/kg) and DES (0.004 or 0.02 mg/kg) on cAMP production of ovarian follicles derived from offspring on PND 14, the freshly isolated follicles were preincubated in the absence of hFSH for 12 h. Thereafter the follicles were incubated without and with 0.11, 1.1 and 11 IU hFSH for 1 h in the presence of 250 mM 3-isobutyl-1-methyl-xantine (IBMX; Sigma). The cAMP levels were analysed by a protein binding assay [23] as described earlier [24]. To study the effect of in vitro TCDD-treatment, follicles isolated from non-treated animals were cultured in the medium containing hFSH (1.1 IU) and TCDD (0, 0.01, 0.1, 1, 10 or 50 nM) for 5 days. At the end of the culture, adenylate cyclase was stimulated with 1 or 10 pM forskolin for 20 min in the presence of 250 M IBMX. Intra- and inter-assay variations were below 12 and 22%, respectively.
2.8. Hormone measurements E2, P4 and T were measured from diethyl ether extracts of heparin plasma and culture media by time-resolved fluoroimmunoassay DELFIA® (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) as described earlier [21]. Serum FSH concentrations were determined by two-site time-resolved immunofluorometric assay, DELFIA® (PerkinElmer Life and Analytical Sciences) for rat FSH [25]. The assay detection limit for E2 was 13.6 pg/ml, for P4 250 pg/ml, for T 100 pg/ml, and for FSH 0.100 ng/ml.
2.9. Western blot analysis Analysis for P4 receptor, StAR, P450scc, 3-HSD and P450arom protein expression in the ovary was performed using two pups (four pooled ovaries) as one sample. Ovaries dissected from control and TCDD-exposed animals were snap frozen in liquid nitrogen and homogenized in lysis buffer (pH 8.0) containing 20 mM Tris, 100 mM NaCl, 2 mM EDTA and 2% Nonidet P40 and supplemented with protease inhibitors phenylmethylsulphonylfluoride (1 mM), aprotinin (1 mM), and leupeptin (1 mM). The lysate was centrifuged at 14,000 rpm for 25 min at 4 ◦ C. Protein concentration in supernatant was measured by the Bradford assay. Proteins (30 g) from control or TCDD-treated animals were electrophorized in 12.5% SDS-PAGE and electroblotted onto nitrocellulose membrane (Protrun, BA 85, Schleicher&Schuell BioScience, Germany). Membranes were blocked overnight for unspecific antibody binding in phosphate-buffered saline containing 3% milk powder and 0.3% Tween-20. The blots were incubated in PBS blocking solution containing anti-P4 receptor (1:20,000), anti-human StAR (1:5000), anti-P450scc (1:5000), anti-3-HSD (1:10,000) or anti-P450arom (1:5000) antibodies for 1 h at RT. Monoclonal
523
mouse anti-P4 receptor antibodies were purchased from Zymed Laboratories (San Francisco, USA). Polyclonal rabbit anti-human StAR antibody was a courtesy of Dr. Jerome F. Strauss III (University of Pennsylvania, Medical Center, USA) and anti-3-HSD antibody a courtesy of Ian Mason (University of Edinburg, England). Polyclonal rabbit anti-rat P450scc (AB1294) was purchased from Chemicon, UK and monoclonal anti-P450arom antibody (MAb3-2C2) from Hauptman-Woodward Institute, New York, USA. Monoclonal anti-actin antibody (Sigma) was used as a protein loading control. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Amersham Life Science, USA), were used for ECL chemiluminescent system labelling (Amersham Pharmatica Biotech, USA). The relative amount of proteins was quantified by densitometry (Chemi ImagerTM , Alpha Innotech Corporation, USA). Data were normalized for -actin levels.
2.10. Statistics All analyses were done using SPSS 9.0.1 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. A p value less than 0.05 was considered as the limit for statistical significance.
3. Results 3.1. Body weight and survival of animals The litter size or the time of delivery were not affected by TCDD at any doses used or by DES at doses 0.004 and 0.02 mg/kg. The postnatal survival of pups was not affected either. However, no live pups were born at the dose 0.1 mg DES/kg. In most of the cases, delivery did not proceed and pregnant dams were euthanized latest on the pregnancy day 23. In utero and lactational exposure to TCDD or to DES had no statistically significant effect on the body weight of female offspring (Table 1). 3.2. Plasma hormones DES had no statistically significant effects on FSH, E2 and P4 levels of female pups on PND 14 (Table 1). In TCDD-treated animals, FSH and E2 levels were not affected, but significant increase (p < 0.01) in P4 levels was seen at a dose 1.0 g TCDD/kg (Table 1).
Table 1 Effects of in utero and lactational exposure to TCDD and DES on body weight and plasma level of follicle stimulating hormone (FSH), estradiol (E2), and progesterone (P4) in female rat offspring on PND 14 Dose TCDDa
DESb
Body weight (g)
FSH (ng/ml)
E2 (pg/ml)
P4 (ng/ml)
Number of litters
0 0.04 0.2 1.0 – – – –
– – – – 0 0.004 0.02 0.1c
33.9 ± 1.6 34.1 ± 1.0 34.6 ± 3.0 31.1 ± 1.2 34.1 ± 2.5 33.7 ± 1.9 35.4 ± 1.2 –
45.7 ± 12.6 44.6 ± 5.9 49.1 ± 4.8 44.6 ± 8.1 33.0 ± 3.6 30.0 ± 6.5 31.4 ± 4.6 –
80.6 ± 8.2 81.1 ± 4.8 78.2 ± 6.9 74.4 ± 12.3 76.0 ± 8.2 80.5 ± 13.5 70.4 ± 9.7 –
0.63 ± 0.16 0.62 ± 0.11 0.56 ± 0.08 0.99 ± 0.11** 1.31 ± 0.23 1.48 ± 0.37 1.33 ± 0.40 –
6 6 6 5 8 6 9 6
Note: Values in each group are mean ± S.D. of litter averages. a Dose of TCDD (g/kg). b Dose of DES (mg/kg). c At dose 0.1 mg/kg, no live pups were born. ** Differs significantly from corresponding control, p < 0.01.
524
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
Fig. 1. Effect of in utero and lactational exposure to TCDD (A) and DES (B) on hFSH-stimulated cAMP production of ovarian follicles isolated from a female rat offspring on PND 14. A 12-h pre-incubation in the absence of hFSH was followed by a 1 h stimulation with hFSH + 250 mM IBMX. Values are mean ± S.D. of 3 (TCDD) or 2 (DES) litter averages. * p < 0.05 treatment vs. control. TCDD doses: control (open circle), 0.04 g/kg (open triangle), 0.2 g/kg (grey triangle), 1.0 g/kg (closed triangle). DES doses: control (open circle), 0.004 mg/kg (open triangle), 0.02 mg/kg (grey triangle).
3.3. Aromatase activity Previously we have shown that aromatase activity of follicles was decreased after maternal TCDD-exposure [7]. In the present study, maternal exposure to DES had no effect on aromatase activity of ovarian follicles derived from female offspring on PND 14 (data not shown). TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM had no effect on P450arom activity of ovarian follicles derived from non-treated animals on PND 14 (data not shown). We have previously shown that DES in vitro did not either affect aromatase activity of isolated ovarian follicles [21]. 3.4. cAMP production In each experiment, follicles isolated from control, TCDD, or DES-exposed animals showed a dose-dependent increase in cAMP levels after hFSH-stimulation. In utero and lactational exposure to TCDD increased hFSH-stimulated cAMP production of isolated ovarian follicles, while basal cAMP production was not affected (Fig. 1A). Statistically significant increase (p < 0.05) was seen with 1.1 IU hFSH at group given 0.2 g TCDD/kg. Maternal exposure to DES had no effect on basal or hFSH-stimulated cAMP production (Fig. 1B). TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM had no effect on basal of hFSH-stimulated cAMP production of ovarian follicles derived from non-treated animals (Table 2). We have previously shown with identical experimental set up that neither DES in vitro at doses of 10 nor 100 nM had an effect on cAMP formation [21]. 3.5. Hormone production
E2 and T secretion was increased significantly (p < 0.001) at the dose 0.02 mg DES/kg. TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM showed no effects on P4, E2 or T production of ovarian follicles derived from non-treated animals (Fig. 4). We have shown earlier that DES in vitro at doses of 10, 100 and 1000 nM decreased dramatically steroid hormone production in 3- and 5-day follicle cultures [21]. 3.6. Ovarian protein expression The protein expression of ovarian P4 receptor, StAR, P450scc, 3-HSD and P450arom was not affected by TCDD in the ovary of 14-day-old Sprague–Dawley rats (Fig. 5). However, relatively large variation between individuals in StAR protein expression was seen both in control and in TCDD-treated groups. 4. Discussion In utero and lactational exposure TCDD had no statistically significant effect on female offspring body weight on PND 14. Neither plasma E2 or gonadotropin levels were changed, whereas P4 level was increased at the TCDD dose 1 g/kg. The increased plasma P4 level in this study in females manifesting inhibited ovarian P450arom activity, as demonstrated in a Table 2 Effect of in vitro TCDD exposure (5 days) on basal and FSH-stimulated cAMP production of follicles isolated from ovaries of a non-treated female rat offspring on PND 14 TCDD (nM)
In 3 and 5 days follicle cultures, follicles isolated from maternally TCDD-exposed animals showed a decreased P4 secretion into the culture medium compared to control follicles (Fig. 2). Statistically significant decrease was seen at all TCDD doses used. The secreted T level was relatively low in each group, and no effects by TCDD were seen. E2 levels were not affected by TCDD after 3 days in culture, but a decreased (p < 0.05) level of E2 was seen at the dose 1.0 g TCDD/kg after a 5-day culture. Maternal exposure to DES had no effect on P4 levels of isolated ovarian follicles in a 3-day culture (Fig. 3). However,
cAMP (fmol/follicle) 0
0 0.01 0.1 1 10 50
5.2 8.4 7.8 9.1 11.1 6.5
± ± ± ± ± ±
3.5 7.1 4.0 6.8 5.0 4.3
0.011
0.11
1.1a
15.6 ± 6.8 12.9 ± 4.9 10.2 ± 5.0 10.3 ± 5.3 12.4 ± 5.8 15.2 ± 4.9
32.5 ± 11.8 34.7 ± 15.2 28.7 ± 6.8 34.7 ± 10.1 29.1 ± 8.2 26.5 ± 8.0
37.4 ± 10.4 35.5 ± 12.6 34.3 ± 13.9 35.8 ± 7.6 38.8 ± 7.1 39.5 ± 6.0
Note: Values are mean ± S.D. of cAMP produced per follicle during 1 h hFSHstimulation. a Dose of hFSH (IU). Number of independent replications in each group, 7–12.
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
525
Fig. 2. Effect of in utero and lactational exposure to TCDD on hormone production of ovarian follicles isolated from a female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle cultures each having 15–50 follicles. For each exposure group, 3 litters were used (2 pups per litter pooled for one follicle isolation). * p < 0.05, ** p < 0.01, *** p < 0.001 treatment vs. corresponding control. n = number of culture plates.
parallel study by Myllym¨aki et al. [7], might be due to the accumulation of early products of the steroidogenic pathway as a consequence of inhibited aromatase enzyme activity. Maternal DES-exposure was used as a treatment control in the present study. In utero exposure to DES at doses of 0.004 and 0.02 mg/kg on gestational days 13, 15 and 17 had no effect on body weight or plasma hormone profile of 14-day-old female pups. The highest dose of DES (0.1 mg/kg) proved to cause a dystocia, and no live pups were delivered by dams in this exposure group. It is proposed that the dystocia experienced by DES-treated dams is directly related to a delay in the onset of labor [17]. In line with the present study, the developmental toxicity of DES after maternal exposure has been described in rats [15,18,26,27]. In line with the present study showing decreased P4 and E2 secretion of cultured ovarian follicles after maternal TCDD exposure, the inhibitory action of maternally introduced TCDD on ovarian P450arom activity and P450arom, P450scc and StAR
mRNA levels was demonstrated earlier [7]. Compared to P4, the effect of TCDD on E2 secretion was relatively minor and evident only after a 5-day follicle culture. This may be explained by a difference in substrate supply for E2 and P4 production. In the absence of exogenous substrate, the majority of endogenous androstenedione could presumably be converted to E2 in spite of decreased P450arom activity. This is evidenced by the studies performed with a low concentration of substrate (1 pM), which showed no differences in the P450arom activity between control and TCDD-exposed follicles ([7], data not shown). When higher concentration of exogenous androstenedione (10 pM) was added to the culture medium to ensure the excess substrate supply for P450arom enzyme, the difference between control and exposed follicles became evident [7]. Although the mechanism of TCDD-induced interference with steroidogenesis is still unclear, it apparently is not related to cAMP formation. hFSH-stimulated ex vivo cAMP levels of follicles were not inhibited by TCDD, and at TCDD dose 0.2 g/kg
Fig. 3. Effect of in utero and lactational exposure to DES on hormone production of ovarian follicles isolated from a female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle cultures each having 15–50 follicles. For control and DES 0.02 mg/kg groups, four litters were used, and for 0.004 mg/kg group, two litters were used (two pups per litter pooled for one follicle isolation). *** p < 0.001 treatment vs. corresponding control. n = number of culture plates.
526
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
Fig. 4. Effect of TCDD in vitro exposure on progesterone (P4), testosterone (T), and estradiol (E2) production of ovarian follicles isolated from ovaries of a nontreated female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle culture plates each having 15–50 follicles.
the maximal cAMP level induced by 1 IU of hFSH was even significantly above the control level. However, the biological relevance of this increase is questionable since it was seen only at the highest dose of hFSH. Furthermore, the possible mechanism behind this cAMP elevation remains unresolved. Hirakawa et al. [28] demonstrated that TCDD-induced interference with FSH action did not involve cAMP generation in cultured granulosa cells. These findings are in line with our previous study showing that maternal TCDD exposure does not inhibit hCGstimulated cAMP levels in 19.5-day-old fetal rat testis [19]. Maternal DES at a dose of 0.02 mg/kg increased significantly hFSH-stimulated ex vivo E2 production of isolated ovarian fol-
licles. Simultaneous increase in T production and no change in P450arom activity suggest that DES primarily stimulates the early steps of steroidogenic pathway. In support of our results, Ghersevich et al. [16] showed in DES-treated rat granulosa cells an increased activity of 17-HSD type 1, an enzyme converting androstenedione to testosterone and estrone to estradiol. Unlike natural estrogens, DES does not bind to ␣-fetoprotein [29], highly expressed steroid binding protein in the blood of 14-day-old rats [30], and DES is therefore suggested to be relatively effective to promote the maturation of ovarian follicles [15]. Although maternal TCDD exposure decreased ovarian mRNA levels of StAR, P450scc and P450arom [7], the protein levels of ovarian StAR, P450scc, 3-HSD, P450arom and P4 receptor were apparently not affected on PND 14 as judged by Western blot analysis. Compared to semi-quantitative PCR method used to measure ovarian mRNA levels, Western blot analysis is an insensitive method and therefore small differences at the protein expression between control and TCDD-treated animals may not be detected. On the other hand, the changes at the protein level may be seen only after a translational delay. Especially for P450scc, the decreased mRNA levels were seen not until on PND 16 [7], and it is therefore possible that the expression at the protein level is not affected on PND 14. Furthermore, the control of gene expression in eukaryotic cells is complex and the changes in mRNA and protein levels do not necessarily correlate. Therefore, the lack of evidence of TCDD-induced changes in the protein expression of ovarian steroidogenic enzymes does not necessarily equal with the lack of effect. Further studies are needed to clarify the effects of these transitional TCDD-induced changes on fertility and reproductive capacity in adulthood. TCDD in vitro at doses of 0.01–50 nM showed no effects on P4, E2, T or cAMP production of immature rat ovarian follicles. Ovarian P450arom activity was not affected by TCDD in vitro exposure either. Although it is complicated to compare tissue concentrations between in vitro and in vivo studies, the highest concentration used in the present study (50 nM = 16.1 ng/nl) is likely to result in clearly higher tissue concentration than the maternal doses used in in vivo studies (0.04–1 g/kg). We recently reported that exposure of pregnant rats to 0.5 g TCDD/kg on gestational day 15 resulted in offspring body burden (average tissue concentration) of 276 pg/g fresh wt on PND 5 [31]. Therefore the results suggest that the effects of TCDD on the immature ovary within a relevant range of concentrations are likely to be indirect, and that the altered metabolism and the activation of detoxification mechanisms may have an essential role in TCDD-mediated disruption of steroidogenesis. In line with our study, Son et al. [32] demonstrated the lack of a direct in vitro effect of TCDD at doses 10–800 nM on whole ovarian dispersate cells of Sprague–Dawley rats. In porcine preovulatory follicle culture, TCDD added to culture medium at a dose 3.2 ng/g tissue accumulated in the tissues after 24 and 96 h incubation by 59.3 and 81.2%, respectively [33]. Therefore, it can be expected that in 24, 72 and 120 h cultures at doses 0.01–50 nM TCDD is bioavailable in the rat ovarian follicles. However, the present lack of effects at environmentally relevant or even at high doses of TCDD suggests that immature rat ovarian follicle
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
527
Fig. 5. Western blot analysis of progesterone (P4) receptor, steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3-hydroxysteroid dehydrogenase (3-HSD), and P450 aromatase (P450arom) protein of freshly isolated ovaries of rat offspring on PND 14 after in utero and lactational exposure to TCDD. Data were normalized for the abundance of -actin. The optical density of the obtained bands was measured by densitometry. Values are mean ± S.D. of 4 or 5 litter averages. Ovaries of two pups (four ovaries) from the same litter are pooled as a one sample.
culture method is not a sensitive model to study the mechanisms of TCDD action. In summary, maternal exposure to DES exhibited a distinct ex vivo stimulation of steroidogenesis in the isolated prepubertal ovarian follicles. The studies confirmed the applicability of the ex vivo follicle culture system for the reproductive toxicology screening in female offspring. The data suggests that maternally introduced TCDD may have farreaching reproductive consequences in female offspring. The strongest inhibition in ex vivo progesterone production of isolated ovarian follicles suggests that TCDD primarily interferes with early phases of ovarian steroidogenic pathway. Furthermore, the inhibitory effect earlier demonstrated in the mRNA expression of ovarian StAR, P450scc and P450arom and in P450arom activity [7], is not apparent at the protein level. TCDD in vitro at doses 0.01–50 nM had no direct effect on follicular steroidogenesis suggesting that inhibition in follicular ex vivo steroid hormone production is an indirect ovarian effect and/or related to TCDD-induced metabolic alterations in vivo. Further research is needed to resolve the putative ovarianspecific mechanisms of TCDD’s action. However, the immature rat ovarian follicle culture method seems not to be a sensitive model to study the mechanisms of TCDD action.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Acknowledgements 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, Sigrid Jus´elius Foundation, the European Commission under the framework of the “Quality of Life” programme (Contracts #: QLK4-CT1999-01422, QLK4-CT1999-01446, QLK4CT2002-00603, and QLK4-CT2002-02528), Turku University Central Hospital, and The Academy of Finland. References [1] Gao X, Mizuyachi K, Terranova PF, Rozman KK. 2,3,7,8Tetrachlorodibenzo-p-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory
[10]
[11]
[12]
[13]
gonadotropin secretion in the immature gonadotropin-primed rat. Toxicol Appl Pharmacol 2001;170:181–90. Li X, Johnson DC, Rozman KK. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases release of luteinizing hormone and follicle-stimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicol Appl Pharmacol 1997;142:264–9. Salisbury TB, Marcinkiewicz JL. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran redices growth and disrupts reproductive parameters in female rats. Biol Reprod 2002;66:1621–6. Chaffin CL, Trewin AL, Watanabe G, Taya K, Hutz RJ. Alterations to the pituitary-gonadal axis in the peripubertal female rat exposed in utero and through lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biol Reprod 1997;56:1498–502. Dasmahapatra AK, Wimpee BAB, Trewin AL, Wimpee CF, Ghorai JK, Hutz RJ. Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay. Mol Cell Endocrinol 2000;164:5–18. Gregoraszczuk EL, Zabielny E, Ochwat D. Aryl hydrocarbon receptor (AhR)-linked inhibition of luteal cell progesterone secretion in 2,3,7,8-tetrachlorodibenzo-p-dioxin treated cells. J Physiol Pharmacol 2001;52:303–11. Myllym¨aki SA, Haavisto TE, Brokken LJS, Viluksela M, Toppari J, Paranko J. In utero and lactational exposure to TCDD; Steroidogenic outcomes differ in male and female rat pups. Toxicol Sci 2005;88:534–44. Gray LE, Ostby JS. In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol Appl Pharmacol 1995;133:285–94. Mably TA, Moore RW, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol Appl Pharmacol 1992;114:97–107. Chen C-Y, Hamm JT, Hass JR, Birnbaum LS. Disposition of polychlorinated dibenzo-p-dioxins, dibenzofurans, and non-ortho polychlorinated biphenyls in pregnant Long Evans rats and the transfer to offspring. Toxicol Appl Pharmacol 2001;173:65–88. Chu I, Villeneuve DC, Yagminas A, et al. Toxicity of PCB 77 (3,3 ,4,4 tetrachlorobiphenyl) and PCB 118 (2,3 ,4,4 ,5-pentachlorobiphenyl) in the rat following subchronic dietary exposure. Fundam Appl Toxicol 1995;26:282–92. Petroff BK, Gao X, Rozman KK, Terranova PF. Interaction of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects. Reprod Toxicol 2000;14:247–55. Petroff BK, Gao X, Rozman KK, Terranova PF. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on weight gain and hepatic ethoxyresorufin-o-deethylase (EROD) induction vary with ovarian hormonal status in the immature gonadotropin-primed rat model. Reprod Toxicol 2001;15:269–74.
528
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
[14] Wyde ME, Eldridge SR, Lucier GW, Walker NJ. Regulation of 2,3,7,8tetrachlorodibenzo-p-dioxin-induced tumor promotion by 17-estradiol in female Sprague–Dawley rats. Toxicol Appl Pharmacol 2001;173:7–17. [15] Yamamoto M, Shirai M, Sugita K, Nagai N, Miura Y, Mogi R, et al. Effects of maternal exposure to diethylstilbestrol on the development of the reproductive system and thyroid function in male and female rat offspring. J Toxicol Sci 2003;28:385–94. [16] Ghersevich S, Poutanen M, Tapanainen J, Vihko R. Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor. Endocrinology 1994;135:1963–71. [17] Zimmerman SA, Clevenger WR, Brimhall BB, Bradshaw WS. Diethylstilbestrol-induced perinatal lethality in the rat. II. Perturbation of parturition. Biol Reprod 1991;44:583–9. [18] Piersma AH, Verhoef A, Sweep CGJ, de Jong WH, van Loveren H. Developmental toxicity but no immunotoxicity in the rat after prenatal exposure to diethylstilbestrol. Toxicology 2002;174:173–81. [19] Haavisto TE, Nurmela K, Pohjanvirta R, Huuskonen H, El Gehani F, Paranko J. Prenatal testosterone and luteinizing hormone levels in male rats exposed during pregnancy to 2,3,7,8-tetrachlorodibenzo-p-dioxin and diethylstilbestrol. Mol Cell Endocrinol 2001;178:169–79. [20] Cain L, Chatterjee S, Collins TJ. In vitro folliculogenesis of rat preantral follicles. Endocrinology 1995;136:3369–77. [21] Myllym¨aki S, Haavisto T, Vainio M, Toppari J, Paranko J. In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tert-octylphenol on steroidogenic activity of isolated immature rat ovarian follicles. Toxicol Appl Phamacol 2005;204:69–80. [22] Lephart ED, Simpson ER. Assay of aromatase activity. Methods Enzymol 1991;206:477–83. [23] Nordstedt C, Fredholm BB. A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid. Anal Biochem 1990;189:231–4. [24] Haavisto TE, Adamsson NA, Myllym¨aki SA, Toppari J, Paranko J. Effects of 4-tert-octylphenol, 4-tert-butylphenol, and diethylstilbestrol on prenatal testosterone surge in the rat. Reprod Toxicol 2003;17:593–605.
[25] van Casteren JL, Schoonen WG, Kloosterboer HJ. Development of timeresolved immunofluorometric assays for rat follicle-stimulating hormone and luteinizing hormone and application on sera of cycling rats. Biol Reprod 2000;62:886–94. [26] Rands PL, White RD, Carter MW, Allen SD, Bradshaw WS. Indicators of fevelopmental toxicity following prenatal administration of hormonally active compounds in the rat. I. Gestational length. Teratology 1982;25:37–43. [27] Wardell RE, Seegmiller RE, Bradshaw WS. Induction of prenatal toxicity in the rat by diethylstilbestrol, 3,4,3 ,4 ,-tetrachlorobiphenyl, cadmium, and lead. Teratology 1982;26:229–37. [28] Hirakawa T, Minegishi T, Abe K, et al. Effect of 2,3,7,8-tetrecholodibenzop-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells. Endocrinology 2000;141:1470–6. [29] Savu L, Benassayag C, Vallette G, Nunez EA. Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1fetoprotein and albumin. Steroids 1979;34:737–48. [30] Meijs-Roelofs HM, Kramer P. Maturation of the inhibitory feedback action of oestrogen on follicle-stimulating hormone secretion in the immature female rat: a role for alpha-foetoprotein. J Endocrinol 1979;81:199– 208. [31] Miettinen HM, Pulkkinen P, J¨ams¨a T, Koistinen J, Simanainen U, Tuomisto J, et al. Effects of in utero and lactational TCDD exposure on bone development in differentially sensitive rat lines. Toxicol Sci 2005;85:1003– 12. [32] Son D, Ushinohama K, Gao X, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reprod Toxicol 1999;13:521–30. [33] Grochowalski A, Pieklo R, Gasinska A, Chrzaszcz R, Gregoraszczuk EL. Accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in porcine preovulatory follicles after in vitro exposure to TCDD: effects on steroid secretion and cell proliferation. Cytobios 2000;102:21– 31.
Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on rat follicular steroidogenesis Sari A. Pesonen a,b,∗ , Tapio E. Haavisto a , Matti Viluksela c , Jorma Toppari b,d , Jorma Paranko e a
Department of Biology, Laboratory of Animal Physiology, University of Turku, 20014 Turku, Finland b Department of Physiology, University of Turku, 20520 Turku, Finland c Department of Environmental Health, Laboratory of Toxicology, National Public Health Institute, Box 95, 70701 Kuopio, Finland d Department of Pediatrics, University of Turku, 20520 Turku, Finland e Department of Anatomy, University of Turku, 20520 Turku, Finland Received 19 December 2005; received in revised form 8 February 2006; accepted 3 March 2006 Available online 18 May 2006
Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental pollutant and causes adverse effects on female reproduction when administered to rats. Our aims were to study effects of gestational and lactational exposure to TCDD on ovarian steroidogenesis and steroidogenic enzyme expression of offspring on postnatal day (PND) 14 in the rat and sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro. Synthetic estrogen diethylstilbestrol (DES) was used as a treatment control. Serum progesterone (P4) level in offspring increased significantly on PND 14 in the TCDD (1 g/kg)-exposed group while body weight, FSH and E2 levels were not changed. In ovarian follicles of offspring on PND 14 in the TCDD-exposed groups, protein expression of cytochrome P-450 aromatase, cytochrome P-450 cholesterol side-chain cleavage, steroidogenic acute regulatory protein, 3-hydroxy-steroid-dehydrogenase/5 -4 isomerase type 1, or P4 receptor was not affected. TCDD decreased E2 and P4 production in ex vivo follicle culture. DES at a dose level of 0.1 mg/kg was dystocic while a dose 0.02 mg/kg increased ovarian ex vivo E2 and testosterone production without affecting P450arom activity indicating stimulation of early steps of steroidogenic pathway. Data suggests that TCDD has multiple targets in ovarian steroidogenesis, but the inhibitory action represented as decreased follicular steroid hormone production ex vivo is not apparent at the ovarian protein expression. Furthermore, TCDD had no direct effect on immature rat ovarian steroidogenesis in vitro suggesting that the follicle culture method is not a sensitive method to study the mechanisms of TCDD action. © 2006 Elsevier Inc. All rights reserved. Keywords: TCDD; DES; Ovary; Follicle; Steroidogenesis; Aromatase; Maternal exposure; Offspring
1. Introduction 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and highly toxic environmental pollutant and it bioaccumulates in the body due to its lipophilic properties and slow metabolism and excretion. There is an increasing evidence of TCDD’s endocrine disrupting effects on immature female reproductive system. In the hypothalamus, TCDD has been shown to decrease the responsiveness to estradiol [1]. A direct stimulatory effect of
∗
Corresponding author at: Department of Physiology, University of Turku, 20520 Turku, Finland. Tel.: +358 50 5730 433; fax: +358 2 3337352. E-mail address: [email protected] (S.A. Pesonen). 0890-6238/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2006.03.007
TCDD on pituitary gonadotropin secretion in immature female rats was demonstrated by Li et al. [2]. In utero and lactational exposure to TCDD has been shown to disrupt estrus cycles and inhibit ovulation rate [3]. Furthermore, maternal exposure to TCDD has potency to directly disturb ovarian steroidogenesis of female progeny [4] and TCDD has been shown to alter the activity and expression of several ovarian steroidogenic enzymes either directly or indirectly [5–7]. Reproductive organs and endocrine system are sensitive to TCDD during development and sexual maturation [8,9]. Compared to placental transfer, exposure via lactation plays a major role in offspring receiving maternal PCDD/Fs and dioxin-like PCBs [10]. Female Sprague–Dawley rats appear to be more sensitive to dioxin-like PCBs than males [11]. It has been proposed,
522
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
that the increased susceptibility of females to TCDD toxicity may be due to the sex-specific kinetics of TCDD [12]. The sensitivity to TCDD has been demonstrated to vary with the alterations in ovarian hormonal status [13], and estrogens have been shown to modulate the effects exerted by TCDD [12,14]. Therefore, the intensive hormone metabolism in the ovary during sexual maturation at the age of 2 weeks may increase the susceptibility of infantile female rat to TCDD. Maternally introduced diethylstilbestrol (DES), was used as a treatment control. Maternal DES exposure has been shown to promote the reproductive functions of immature female rat by increasing plasma FSH concentration and by accelerating the development of primary and secondary follicles in the ovary [15]. In cultured ovarian cells, stimulatory effect of DES has been demonstrated by increased 17-HSD-activity, an enzyme converting androstenedione to testosterone and estrone to estradiol [16]. Also developmental toxicity of DES in terms of dystocia and decreased pup body weight has been reported [17,18]. The mechanism(s) of TCDD-induced alterations in the ovary are still unclear. Furthermore, little is known about low-dose effects of TCDD on immature ovarian steroidogenesis. We have previously demonstrated that in immature rat ovary, maternal TCDD decreases cytochrome P-450 aromatase enzyme (P450arom) activity, and mRNA levels of P450arom, cytochrome P-450 cholesterol side-chain cleavage (P450scc), and steroidogenic acute regulatory protein (StAR) [7]. In the present study, we studied effects of in utero and lactational exposure to TCDD on ovarian steroidogenesis ex vivo and protein expression of ovarian steroidogenic enzymes in offspring on postnatal day (PND) 14 in the rat. Furthermore, the sensitivity of enzymatically isolated ovarian follicles to TCDD in vitro was studied. 2. Materials ad methods 2.1. Animals Timed mated Sprague–Dawley rats (Harlan, Zeist, The Netherlands) 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 0. Rats were housed individually in plastic cages with wire-mesh covers in a room with a 12-h light/12-h 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 tap water and standard pelleted laboratory animal feed R36 (Ewos, S¨odertelje, Sweden) in TCDD-exposures and Commercial RM1 (E) SQC (Special Diet Services, Witham, England) in DES-exposures, respectively. The day of birth was considered postnatal day (PND) 0. One day after birth the litter size was adjusted to eight pups by random culling to allow uniform lactational exposure. Dams were allowed to nurse offspring until the termination of the experiment on PND 14. The Animal Experiment Committee of the University of Kuopio and the Kuopio Provincial Government, and The Turku University Committee on Ethics of Animal Experimentation approved the experimental protocols.
2.2. TCDD treatments TCDD (Ufa-Oil Institute, Ufa, 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 let to evaporate. Dosing solutions were carefully mixed in a magnetic stirrer and sonicated for 20 min before dosing. Diethyl ether and corn oil were of analyt-
ical grade and purchased from Merck (Darmstadt, Germany) and from BDH Laboratory Supplies (Poole, England), respectively. A single maternal dose of TCDD (0.04, 0.2 or 1.0 g/kg) was given in 4 ml/kg corn oil by gavage on gestational day 13. Controls were given corn oil alone. Dose levels and the timing of exposure were similar to our previous parallel study showing that identical TCDD-exposure inhibited immature rat ovarian P450arom activity and mRNA levels of P450arom, P450scc, and StAR protein [7]. In the present study, we wanted to further study TCDD-induced ovarian steroidogenic inhibition.
2.3. DES treatments DES (Sigma, St. Louis, USA) was dissolved in dimethylsulphoxide (DMSO; Sigma) and adjusted volumes of the solutions were mixed with corn oil. Total of three subcutaneous injections of DES (0.004, 0.02 or 0.1 mg/kg) were given in the volume of 300 l corn oil on gestational days 13, 15 and 17. Controls were given DMSO in corn oil alone. In our earlier study, identical DES-exposure at a dose of 0.1 mg/kg decreased significantly testicular and plasma T content of male rat fetuses [19]. In this study, the highest dose level and the days for treatment were selected to study whether DES-induced steroidogenic disruption could be seen also in female gonads.
2.4. Sampling On the PND 14, individual body weight was recorded and pups were euthanized by cervical dislocation after mixture of carbon dioxide and oxygen anesthesia. Blood was obtained by cardiac puncture into heparinized syringes, kept on ice, and centrifuged for 5 min at 1000 × g at +4 ◦ C. Plasma was stored at −20 ◦ C before measurement of progesterone (P4), estradiol (E2), LH and FSH. Ovaries for Western blot were excised, snap frozen in liquid nitrogen, and stored at −70 ◦ C.
2.5. Follicle culture On the PND 14, ovaries were aseptically collected from offspring euthanized (see above) and given into sterile phosphate-buffered saline (PBS, pH 7.4) solution at RT. Follicles were isolated using the method of Cain et al. [20] with minor modifications described earlier [21]. Follicles, 15–50 for each tissue culture dish (Nunc, Roskilde, Denmark; 1.9 cm2 ) at final volume of 250 l were cultured in medium (McCoy’s 5A, without phenol red and sodium; Sigma) supplemented with 600 mg glucose, 1 ml penicillin–streptomycin, 200 l insulin, 500 g transferrin and 4 g hydrocortisone for each 100 ml of medium under humidified air containing 5% CO2 at 37 ◦ C. Human recombinant follicle-stimulating hormone (hFSH, courtesy of N.V. Organon, The Netherlands) was added to the well at the final concentration of 1.1 IU/250 l. On day 3, a sample of 100 l was taken from the medium and replaced with an equal volume of fresh medium. On day 5 at the end of the experiment, medium was collected and frozen at −20 ◦ C for analysis of E2, P4, and T. For TCDD (0, 0.01, 0.1, 1, 10 or 50 nM) and DES (100 ng/ml = 0.37 M) in vitro studies, the chemical was dissolved in DMSO (Sigma) and added to the culture medium in the beginning of the culture. The final concentration of DMSO in the culture medium was 1 l/ml both in DEStreated and control follicles. The viability of follicular cells was assayed at the end of the culture on the basis of tissue organization and with trypan-blue exclusion method as described earlier [21].
2.6. Aromatase assay P450arom activity was assayed by measuring the incorporation of tritium from [1,2-3 H]androstenedione (NEN, Zaventem, Belgium) into the water phase as described [22]. The in vitro effect of TCDD on ovarian P450arom activity was measured on PND 14 in follicles derived from female offspring whose mothers were not treated. Isolated follicles were cultured for 5 days in the presence of 1.1 IU of hFSH and TCDD at dose 0, 0.01, 0.1, 1, 10 or 50 nM. To study the effect of maternal DES exposure on ovarian P450arom activity of female pups, follicles isolated from control and DES-exposed (0.02 mg/kg) animals were cultured for 5 days in the presence of 1.1 IU of hFSH. On day 4, 10 pM of [1,2-3 H]-labelled androstenedione was added in both studies. After ether extraction of the medium, radioactivity of the water phase was measured
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528 using a Microbeta scintillation counter (Perkin-Elmer Wallac, Turku, Finland). For previously measured P450arom activity of ovarian follicles after maternal TCDD-exposure and in vitro DES treatment, see Section 3.
2.7. cAMP assay To measure the effects of gestational and lactational exposure to TCDD (0.04, 0.2 or 1.0 g/kg) and DES (0.004 or 0.02 mg/kg) on cAMP production of ovarian follicles derived from offspring on PND 14, the freshly isolated follicles were preincubated in the absence of hFSH for 12 h. Thereafter the follicles were incubated without and with 0.11, 1.1 and 11 IU hFSH for 1 h in the presence of 250 mM 3-isobutyl-1-methyl-xantine (IBMX; Sigma). The cAMP levels were analysed by a protein binding assay [23] as described earlier [24]. To study the effect of in vitro TCDD-treatment, follicles isolated from non-treated animals were cultured in the medium containing hFSH (1.1 IU) and TCDD (0, 0.01, 0.1, 1, 10 or 50 nM) for 5 days. At the end of the culture, adenylate cyclase was stimulated with 1 or 10 pM forskolin for 20 min in the presence of 250 M IBMX. Intra- and inter-assay variations were below 12 and 22%, respectively.
2.8. Hormone measurements E2, P4 and T were measured from diethyl ether extracts of heparin plasma and culture media by time-resolved fluoroimmunoassay DELFIA® (PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku, Finland) as described earlier [21]. Serum FSH concentrations were determined by two-site time-resolved immunofluorometric assay, DELFIA® (PerkinElmer Life and Analytical Sciences) for rat FSH [25]. The assay detection limit for E2 was 13.6 pg/ml, for P4 250 pg/ml, for T 100 pg/ml, and for FSH 0.100 ng/ml.
2.9. Western blot analysis Analysis for P4 receptor, StAR, P450scc, 3-HSD and P450arom protein expression in the ovary was performed using two pups (four pooled ovaries) as one sample. Ovaries dissected from control and TCDD-exposed animals were snap frozen in liquid nitrogen and homogenized in lysis buffer (pH 8.0) containing 20 mM Tris, 100 mM NaCl, 2 mM EDTA and 2% Nonidet P40 and supplemented with protease inhibitors phenylmethylsulphonylfluoride (1 mM), aprotinin (1 mM), and leupeptin (1 mM). The lysate was centrifuged at 14,000 rpm for 25 min at 4 ◦ C. Protein concentration in supernatant was measured by the Bradford assay. Proteins (30 g) from control or TCDD-treated animals were electrophorized in 12.5% SDS-PAGE and electroblotted onto nitrocellulose membrane (Protrun, BA 85, Schleicher&Schuell BioScience, Germany). Membranes were blocked overnight for unspecific antibody binding in phosphate-buffered saline containing 3% milk powder and 0.3% Tween-20. The blots were incubated in PBS blocking solution containing anti-P4 receptor (1:20,000), anti-human StAR (1:5000), anti-P450scc (1:5000), anti-3-HSD (1:10,000) or anti-P450arom (1:5000) antibodies for 1 h at RT. Monoclonal
523
mouse anti-P4 receptor antibodies were purchased from Zymed Laboratories (San Francisco, USA). Polyclonal rabbit anti-human StAR antibody was a courtesy of Dr. Jerome F. Strauss III (University of Pennsylvania, Medical Center, USA) and anti-3-HSD antibody a courtesy of Ian Mason (University of Edinburg, England). Polyclonal rabbit anti-rat P450scc (AB1294) was purchased from Chemicon, UK and monoclonal anti-P450arom antibody (MAb3-2C2) from Hauptman-Woodward Institute, New York, USA. Monoclonal anti-actin antibody (Sigma) was used as a protein loading control. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Amersham Life Science, USA), were used for ECL chemiluminescent system labelling (Amersham Pharmatica Biotech, USA). The relative amount of proteins was quantified by densitometry (Chemi ImagerTM , Alpha Innotech Corporation, USA). Data were normalized for -actin levels.
2.10. Statistics All analyses were done using SPSS 9.0.1 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. A p value less than 0.05 was considered as the limit for statistical significance.
3. Results 3.1. Body weight and survival of animals The litter size or the time of delivery were not affected by TCDD at any doses used or by DES at doses 0.004 and 0.02 mg/kg. The postnatal survival of pups was not affected either. However, no live pups were born at the dose 0.1 mg DES/kg. In most of the cases, delivery did not proceed and pregnant dams were euthanized latest on the pregnancy day 23. In utero and lactational exposure to TCDD or to DES had no statistically significant effect on the body weight of female offspring (Table 1). 3.2. Plasma hormones DES had no statistically significant effects on FSH, E2 and P4 levels of female pups on PND 14 (Table 1). In TCDD-treated animals, FSH and E2 levels were not affected, but significant increase (p < 0.01) in P4 levels was seen at a dose 1.0 g TCDD/kg (Table 1).
Table 1 Effects of in utero and lactational exposure to TCDD and DES on body weight and plasma level of follicle stimulating hormone (FSH), estradiol (E2), and progesterone (P4) in female rat offspring on PND 14 Dose TCDDa
DESb
Body weight (g)
FSH (ng/ml)
E2 (pg/ml)
P4 (ng/ml)
Number of litters
0 0.04 0.2 1.0 – – – –
– – – – 0 0.004 0.02 0.1c
33.9 ± 1.6 34.1 ± 1.0 34.6 ± 3.0 31.1 ± 1.2 34.1 ± 2.5 33.7 ± 1.9 35.4 ± 1.2 –
45.7 ± 12.6 44.6 ± 5.9 49.1 ± 4.8 44.6 ± 8.1 33.0 ± 3.6 30.0 ± 6.5 31.4 ± 4.6 –
80.6 ± 8.2 81.1 ± 4.8 78.2 ± 6.9 74.4 ± 12.3 76.0 ± 8.2 80.5 ± 13.5 70.4 ± 9.7 –
0.63 ± 0.16 0.62 ± 0.11 0.56 ± 0.08 0.99 ± 0.11** 1.31 ± 0.23 1.48 ± 0.37 1.33 ± 0.40 –
6 6 6 5 8 6 9 6
Note: Values in each group are mean ± S.D. of litter averages. a Dose of TCDD (g/kg). b Dose of DES (mg/kg). c At dose 0.1 mg/kg, no live pups were born. ** Differs significantly from corresponding control, p < 0.01.
524
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
Fig. 1. Effect of in utero and lactational exposure to TCDD (A) and DES (B) on hFSH-stimulated cAMP production of ovarian follicles isolated from a female rat offspring on PND 14. A 12-h pre-incubation in the absence of hFSH was followed by a 1 h stimulation with hFSH + 250 mM IBMX. Values are mean ± S.D. of 3 (TCDD) or 2 (DES) litter averages. * p < 0.05 treatment vs. control. TCDD doses: control (open circle), 0.04 g/kg (open triangle), 0.2 g/kg (grey triangle), 1.0 g/kg (closed triangle). DES doses: control (open circle), 0.004 mg/kg (open triangle), 0.02 mg/kg (grey triangle).
3.3. Aromatase activity Previously we have shown that aromatase activity of follicles was decreased after maternal TCDD-exposure [7]. In the present study, maternal exposure to DES had no effect on aromatase activity of ovarian follicles derived from female offspring on PND 14 (data not shown). TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM had no effect on P450arom activity of ovarian follicles derived from non-treated animals on PND 14 (data not shown). We have previously shown that DES in vitro did not either affect aromatase activity of isolated ovarian follicles [21]. 3.4. cAMP production In each experiment, follicles isolated from control, TCDD, or DES-exposed animals showed a dose-dependent increase in cAMP levels after hFSH-stimulation. In utero and lactational exposure to TCDD increased hFSH-stimulated cAMP production of isolated ovarian follicles, while basal cAMP production was not affected (Fig. 1A). Statistically significant increase (p < 0.05) was seen with 1.1 IU hFSH at group given 0.2 g TCDD/kg. Maternal exposure to DES had no effect on basal or hFSH-stimulated cAMP production (Fig. 1B). TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM had no effect on basal of hFSH-stimulated cAMP production of ovarian follicles derived from non-treated animals (Table 2). We have previously shown with identical experimental set up that neither DES in vitro at doses of 10 nor 100 nM had an effect on cAMP formation [21]. 3.5. Hormone production
E2 and T secretion was increased significantly (p < 0.001) at the dose 0.02 mg DES/kg. TCDD in vitro at doses of 0, 0.01, 0.1, 1, 10 or 50 nM showed no effects on P4, E2 or T production of ovarian follicles derived from non-treated animals (Fig. 4). We have shown earlier that DES in vitro at doses of 10, 100 and 1000 nM decreased dramatically steroid hormone production in 3- and 5-day follicle cultures [21]. 3.6. Ovarian protein expression The protein expression of ovarian P4 receptor, StAR, P450scc, 3-HSD and P450arom was not affected by TCDD in the ovary of 14-day-old Sprague–Dawley rats (Fig. 5). However, relatively large variation between individuals in StAR protein expression was seen both in control and in TCDD-treated groups. 4. Discussion In utero and lactational exposure TCDD had no statistically significant effect on female offspring body weight on PND 14. Neither plasma E2 or gonadotropin levels were changed, whereas P4 level was increased at the TCDD dose 1 g/kg. The increased plasma P4 level in this study in females manifesting inhibited ovarian P450arom activity, as demonstrated in a Table 2 Effect of in vitro TCDD exposure (5 days) on basal and FSH-stimulated cAMP production of follicles isolated from ovaries of a non-treated female rat offspring on PND 14 TCDD (nM)
In 3 and 5 days follicle cultures, follicles isolated from maternally TCDD-exposed animals showed a decreased P4 secretion into the culture medium compared to control follicles (Fig. 2). Statistically significant decrease was seen at all TCDD doses used. The secreted T level was relatively low in each group, and no effects by TCDD were seen. E2 levels were not affected by TCDD after 3 days in culture, but a decreased (p < 0.05) level of E2 was seen at the dose 1.0 g TCDD/kg after a 5-day culture. Maternal exposure to DES had no effect on P4 levels of isolated ovarian follicles in a 3-day culture (Fig. 3). However,
cAMP (fmol/follicle) 0
0 0.01 0.1 1 10 50
5.2 8.4 7.8 9.1 11.1 6.5
± ± ± ± ± ±
3.5 7.1 4.0 6.8 5.0 4.3
0.011
0.11
1.1a
15.6 ± 6.8 12.9 ± 4.9 10.2 ± 5.0 10.3 ± 5.3 12.4 ± 5.8 15.2 ± 4.9
32.5 ± 11.8 34.7 ± 15.2 28.7 ± 6.8 34.7 ± 10.1 29.1 ± 8.2 26.5 ± 8.0
37.4 ± 10.4 35.5 ± 12.6 34.3 ± 13.9 35.8 ± 7.6 38.8 ± 7.1 39.5 ± 6.0
Note: Values are mean ± S.D. of cAMP produced per follicle during 1 h hFSHstimulation. a Dose of hFSH (IU). Number of independent replications in each group, 7–12.
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
525
Fig. 2. Effect of in utero and lactational exposure to TCDD on hormone production of ovarian follicles isolated from a female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle cultures each having 15–50 follicles. For each exposure group, 3 litters were used (2 pups per litter pooled for one follicle isolation). * p < 0.05, ** p < 0.01, *** p < 0.001 treatment vs. corresponding control. n = number of culture plates.
parallel study by Myllym¨aki et al. [7], might be due to the accumulation of early products of the steroidogenic pathway as a consequence of inhibited aromatase enzyme activity. Maternal DES-exposure was used as a treatment control in the present study. In utero exposure to DES at doses of 0.004 and 0.02 mg/kg on gestational days 13, 15 and 17 had no effect on body weight or plasma hormone profile of 14-day-old female pups. The highest dose of DES (0.1 mg/kg) proved to cause a dystocia, and no live pups were delivered by dams in this exposure group. It is proposed that the dystocia experienced by DES-treated dams is directly related to a delay in the onset of labor [17]. In line with the present study, the developmental toxicity of DES after maternal exposure has been described in rats [15,18,26,27]. In line with the present study showing decreased P4 and E2 secretion of cultured ovarian follicles after maternal TCDD exposure, the inhibitory action of maternally introduced TCDD on ovarian P450arom activity and P450arom, P450scc and StAR
mRNA levels was demonstrated earlier [7]. Compared to P4, the effect of TCDD on E2 secretion was relatively minor and evident only after a 5-day follicle culture. This may be explained by a difference in substrate supply for E2 and P4 production. In the absence of exogenous substrate, the majority of endogenous androstenedione could presumably be converted to E2 in spite of decreased P450arom activity. This is evidenced by the studies performed with a low concentration of substrate (1 pM), which showed no differences in the P450arom activity between control and TCDD-exposed follicles ([7], data not shown). When higher concentration of exogenous androstenedione (10 pM) was added to the culture medium to ensure the excess substrate supply for P450arom enzyme, the difference between control and exposed follicles became evident [7]. Although the mechanism of TCDD-induced interference with steroidogenesis is still unclear, it apparently is not related to cAMP formation. hFSH-stimulated ex vivo cAMP levels of follicles were not inhibited by TCDD, and at TCDD dose 0.2 g/kg
Fig. 3. Effect of in utero and lactational exposure to DES on hormone production of ovarian follicles isolated from a female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle cultures each having 15–50 follicles. For control and DES 0.02 mg/kg groups, four litters were used, and for 0.004 mg/kg group, two litters were used (two pups per litter pooled for one follicle isolation). *** p < 0.001 treatment vs. corresponding control. n = number of culture plates.
526
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
Fig. 4. Effect of TCDD in vitro exposure on progesterone (P4), testosterone (T), and estradiol (E2) production of ovarian follicles isolated from ovaries of a nontreated female rat offspring on PND 14. Values are mean ± S.E.M. of separate follicle culture plates each having 15–50 follicles.
the maximal cAMP level induced by 1 IU of hFSH was even significantly above the control level. However, the biological relevance of this increase is questionable since it was seen only at the highest dose of hFSH. Furthermore, the possible mechanism behind this cAMP elevation remains unresolved. Hirakawa et al. [28] demonstrated that TCDD-induced interference with FSH action did not involve cAMP generation in cultured granulosa cells. These findings are in line with our previous study showing that maternal TCDD exposure does not inhibit hCGstimulated cAMP levels in 19.5-day-old fetal rat testis [19]. Maternal DES at a dose of 0.02 mg/kg increased significantly hFSH-stimulated ex vivo E2 production of isolated ovarian fol-
licles. Simultaneous increase in T production and no change in P450arom activity suggest that DES primarily stimulates the early steps of steroidogenic pathway. In support of our results, Ghersevich et al. [16] showed in DES-treated rat granulosa cells an increased activity of 17-HSD type 1, an enzyme converting androstenedione to testosterone and estrone to estradiol. Unlike natural estrogens, DES does not bind to ␣-fetoprotein [29], highly expressed steroid binding protein in the blood of 14-day-old rats [30], and DES is therefore suggested to be relatively effective to promote the maturation of ovarian follicles [15]. Although maternal TCDD exposure decreased ovarian mRNA levels of StAR, P450scc and P450arom [7], the protein levels of ovarian StAR, P450scc, 3-HSD, P450arom and P4 receptor were apparently not affected on PND 14 as judged by Western blot analysis. Compared to semi-quantitative PCR method used to measure ovarian mRNA levels, Western blot analysis is an insensitive method and therefore small differences at the protein expression between control and TCDD-treated animals may not be detected. On the other hand, the changes at the protein level may be seen only after a translational delay. Especially for P450scc, the decreased mRNA levels were seen not until on PND 16 [7], and it is therefore possible that the expression at the protein level is not affected on PND 14. Furthermore, the control of gene expression in eukaryotic cells is complex and the changes in mRNA and protein levels do not necessarily correlate. Therefore, the lack of evidence of TCDD-induced changes in the protein expression of ovarian steroidogenic enzymes does not necessarily equal with the lack of effect. Further studies are needed to clarify the effects of these transitional TCDD-induced changes on fertility and reproductive capacity in adulthood. TCDD in vitro at doses of 0.01–50 nM showed no effects on P4, E2, T or cAMP production of immature rat ovarian follicles. Ovarian P450arom activity was not affected by TCDD in vitro exposure either. Although it is complicated to compare tissue concentrations between in vitro and in vivo studies, the highest concentration used in the present study (50 nM = 16.1 ng/nl) is likely to result in clearly higher tissue concentration than the maternal doses used in in vivo studies (0.04–1 g/kg). We recently reported that exposure of pregnant rats to 0.5 g TCDD/kg on gestational day 15 resulted in offspring body burden (average tissue concentration) of 276 pg/g fresh wt on PND 5 [31]. Therefore the results suggest that the effects of TCDD on the immature ovary within a relevant range of concentrations are likely to be indirect, and that the altered metabolism and the activation of detoxification mechanisms may have an essential role in TCDD-mediated disruption of steroidogenesis. In line with our study, Son et al. [32] demonstrated the lack of a direct in vitro effect of TCDD at doses 10–800 nM on whole ovarian dispersate cells of Sprague–Dawley rats. In porcine preovulatory follicle culture, TCDD added to culture medium at a dose 3.2 ng/g tissue accumulated in the tissues after 24 and 96 h incubation by 59.3 and 81.2%, respectively [33]. Therefore, it can be expected that in 24, 72 and 120 h cultures at doses 0.01–50 nM TCDD is bioavailable in the rat ovarian follicles. However, the present lack of effects at environmentally relevant or even at high doses of TCDD suggests that immature rat ovarian follicle
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
527
Fig. 5. Western blot analysis of progesterone (P4) receptor, steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3-hydroxysteroid dehydrogenase (3-HSD), and P450 aromatase (P450arom) protein of freshly isolated ovaries of rat offspring on PND 14 after in utero and lactational exposure to TCDD. Data were normalized for the abundance of -actin. The optical density of the obtained bands was measured by densitometry. Values are mean ± S.D. of 4 or 5 litter averages. Ovaries of two pups (four ovaries) from the same litter are pooled as a one sample.
culture method is not a sensitive model to study the mechanisms of TCDD action. In summary, maternal exposure to DES exhibited a distinct ex vivo stimulation of steroidogenesis in the isolated prepubertal ovarian follicles. The studies confirmed the applicability of the ex vivo follicle culture system for the reproductive toxicology screening in female offspring. The data suggests that maternally introduced TCDD may have farreaching reproductive consequences in female offspring. The strongest inhibition in ex vivo progesterone production of isolated ovarian follicles suggests that TCDD primarily interferes with early phases of ovarian steroidogenic pathway. Furthermore, the inhibitory effect earlier demonstrated in the mRNA expression of ovarian StAR, P450scc and P450arom and in P450arom activity [7], is not apparent at the protein level. TCDD in vitro at doses 0.01–50 nM had no direct effect on follicular steroidogenesis suggesting that inhibition in follicular ex vivo steroid hormone production is an indirect ovarian effect and/or related to TCDD-induced metabolic alterations in vivo. Further research is needed to resolve the putative ovarianspecific mechanisms of TCDD’s action. However, the immature rat ovarian follicle culture method seems not to be a sensitive model to study the mechanisms of TCDD action.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Acknowledgements 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, Sigrid Jus´elius Foundation, the European Commission under the framework of the “Quality of Life” programme (Contracts #: QLK4-CT1999-01422, QLK4-CT1999-01446, QLK4CT2002-00603, and QLK4-CT2002-02528), Turku University Central Hospital, and The Academy of Finland. References [1] Gao X, Mizuyachi K, Terranova PF, Rozman KK. 2,3,7,8Tetrachlorodibenzo-p-dioxin decreases responsiveness of the hypothalamus to estradiol as a feedback inducer of preovulatory
[10]
[11]
[12]
[13]
gonadotropin secretion in the immature gonadotropin-primed rat. Toxicol Appl Pharmacol 2001;170:181–90. Li X, Johnson DC, Rozman KK. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) increases release of luteinizing hormone and follicle-stimulating hormone from the pituitary of immature female rats in vivo and in vitro. Toxicol Appl Pharmacol 1997;142:264–9. Salisbury TB, Marcinkiewicz JL. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,4,7,8-pentachlorodibenzofuran redices growth and disrupts reproductive parameters in female rats. Biol Reprod 2002;66:1621–6. Chaffin CL, Trewin AL, Watanabe G, Taya K, Hutz RJ. Alterations to the pituitary-gonadal axis in the peripubertal female rat exposed in utero and through lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biol Reprod 1997;56:1498–502. Dasmahapatra AK, Wimpee BAB, Trewin AL, Wimpee CF, Ghorai JK, Hutz RJ. Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by competitive reverse transcriptase-polymerase chain reaction assay. Mol Cell Endocrinol 2000;164:5–18. Gregoraszczuk EL, Zabielny E, Ochwat D. Aryl hydrocarbon receptor (AhR)-linked inhibition of luteal cell progesterone secretion in 2,3,7,8-tetrachlorodibenzo-p-dioxin treated cells. J Physiol Pharmacol 2001;52:303–11. Myllym¨aki SA, Haavisto TE, Brokken LJS, Viluksela M, Toppari J, Paranko J. In utero and lactational exposure to TCDD; Steroidogenic outcomes differ in male and female rat pups. Toxicol Sci 2005;88:534–44. Gray LE, Ostby JS. In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol Appl Pharmacol 1995;133:285–94. Mably TA, Moore RW, Peterson RE. In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol Appl Pharmacol 1992;114:97–107. Chen C-Y, Hamm JT, Hass JR, Birnbaum LS. Disposition of polychlorinated dibenzo-p-dioxins, dibenzofurans, and non-ortho polychlorinated biphenyls in pregnant Long Evans rats and the transfer to offspring. Toxicol Appl Pharmacol 2001;173:65–88. Chu I, Villeneuve DC, Yagminas A, et al. Toxicity of PCB 77 (3,3 ,4,4 tetrachlorobiphenyl) and PCB 118 (2,3 ,4,4 ,5-pentachlorobiphenyl) in the rat following subchronic dietary exposure. Fundam Appl Toxicol 1995;26:282–92. Petroff BK, Gao X, Rozman KK, Terranova PF. Interaction of estradiol and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an ovulation model: evidence for systemic potentiation and local ovarian effects. Reprod Toxicol 2000;14:247–55. Petroff BK, Gao X, Rozman KK, Terranova PF. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on weight gain and hepatic ethoxyresorufin-o-deethylase (EROD) induction vary with ovarian hormonal status in the immature gonadotropin-primed rat model. Reprod Toxicol 2001;15:269–74.
528
S.A. Pesonen et al. / Reproductive Toxicology 22 (2006) 521–528
[14] Wyde ME, Eldridge SR, Lucier GW, Walker NJ. Regulation of 2,3,7,8tetrachlorodibenzo-p-dioxin-induced tumor promotion by 17-estradiol in female Sprague–Dawley rats. Toxicol Appl Pharmacol 2001;173:7–17. [15] Yamamoto M, Shirai M, Sugita K, Nagai N, Miura Y, Mogi R, et al. Effects of maternal exposure to diethylstilbestrol on the development of the reproductive system and thyroid function in male and female rat offspring. J Toxicol Sci 2003;28:385–94. [16] Ghersevich S, Poutanen M, Tapanainen J, Vihko R. Hormonal regulation of rat 17 beta-hydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor. Endocrinology 1994;135:1963–71. [17] Zimmerman SA, Clevenger WR, Brimhall BB, Bradshaw WS. Diethylstilbestrol-induced perinatal lethality in the rat. II. Perturbation of parturition. Biol Reprod 1991;44:583–9. [18] Piersma AH, Verhoef A, Sweep CGJ, de Jong WH, van Loveren H. Developmental toxicity but no immunotoxicity in the rat after prenatal exposure to diethylstilbestrol. Toxicology 2002;174:173–81. [19] Haavisto TE, Nurmela K, Pohjanvirta R, Huuskonen H, El Gehani F, Paranko J. Prenatal testosterone and luteinizing hormone levels in male rats exposed during pregnancy to 2,3,7,8-tetrachlorodibenzo-p-dioxin and diethylstilbestrol. Mol Cell Endocrinol 2001;178:169–79. [20] Cain L, Chatterjee S, Collins TJ. In vitro folliculogenesis of rat preantral follicles. Endocrinology 1995;136:3369–77. [21] Myllym¨aki S, Haavisto T, Vainio M, Toppari J, Paranko J. In vitro effects of diethylstilbestrol, genistein, 4-tert-butylphenol, and 4-tert-octylphenol on steroidogenic activity of isolated immature rat ovarian follicles. Toxicol Appl Phamacol 2005;204:69–80. [22] Lephart ED, Simpson ER. Assay of aromatase activity. Methods Enzymol 1991;206:477–83. [23] Nordstedt C, Fredholm BB. A modification of a protein-binding method for rapid quantification of cAMP in cell-culture supernatants and body fluid. Anal Biochem 1990;189:231–4. [24] Haavisto TE, Adamsson NA, Myllym¨aki SA, Toppari J, Paranko J. Effects of 4-tert-octylphenol, 4-tert-butylphenol, and diethylstilbestrol on prenatal testosterone surge in the rat. Reprod Toxicol 2003;17:593–605.
[25] van Casteren JL, Schoonen WG, Kloosterboer HJ. Development of timeresolved immunofluorometric assays for rat follicle-stimulating hormone and luteinizing hormone and application on sera of cycling rats. Biol Reprod 2000;62:886–94. [26] Rands PL, White RD, Carter MW, Allen SD, Bradshaw WS. Indicators of fevelopmental toxicity following prenatal administration of hormonally active compounds in the rat. I. Gestational length. Teratology 1982;25:37–43. [27] Wardell RE, Seegmiller RE, Bradshaw WS. Induction of prenatal toxicity in the rat by diethylstilbestrol, 3,4,3 ,4 ,-tetrachlorobiphenyl, cadmium, and lead. Teratology 1982;26:229–37. [28] Hirakawa T, Minegishi T, Abe K, et al. Effect of 2,3,7,8-tetrecholodibenzop-dioxin on the expression of follicle-stimulating hormone receptors during cell differentiation in cultured granulosa cells. Endocrinology 2000;141:1470–6. [29] Savu L, Benassayag C, Vallette G, Nunez EA. Ligand properties of diethylstilbestrol: studies with purified native and fatty acid-free rat alpha 1fetoprotein and albumin. Steroids 1979;34:737–48. [30] Meijs-Roelofs HM, Kramer P. Maturation of the inhibitory feedback action of oestrogen on follicle-stimulating hormone secretion in the immature female rat: a role for alpha-foetoprotein. J Endocrinol 1979;81:199– 208. [31] Miettinen HM, Pulkkinen P, J¨ams¨a T, Koistinen J, Simanainen U, Tuomisto J, et al. Effects of in utero and lactational TCDD exposure on bone development in differentially sensitive rat lines. Toxicol Sci 2005;85:1003– 12. [32] Son D, Ushinohama K, Gao X, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro. Reprod Toxicol 1999;13:521–30. [33] Grochowalski A, Pieklo R, Gasinska A, Chrzaszcz R, Gregoraszczuk EL. Accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in porcine preovulatory follicles after in vitro exposure to TCDD: effects on steroid secretion and cell proliferation. Cytobios 2000;102:21– 31.