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Toxicology |
Center for Reproductive Sciences3
Departments of Molecular and Integrative Physiology4 and
Toxicology, Pharmacology, and Experimental Therapeutics,5 University of Kansas Medical Center, Kansas City, Kansas 66160
Physiology and Biophysics,6 University of Colorado School of Medicine, Denver, Colorado 80220
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle-stimulating hormone, gonadotropin-releasing hormone, hypothalamus, luteinizing hormone, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TCDD modulates the response to estrogen for several endpoints in other tissues [6–8], suggesting that alteration of estradiol (E2) feedback to the hypothalamus and pituitary gland is one possible mechanism of dioxin action. Previous studies showed that administration of exogenous E2 during the later stages of synchronized follicular development and ovulation restored a normal gonadotropin surge to equine chorionic gonadotropin (eCG) primed immature rats treated with anovulatory doses of TCDD [9]. This same work demonstrated that while TCDD did not alter serum concentrations of E2 prior to ovulation, the dioxin decreased the responsiveness of the hypothalamus to the positive-feedback effects of E2 during the periovulatory period such that an 8-fold increase in serum E2 was required to restore a normal preovulatory gonadotropin surge in the rat. The role of modulation of the estrogen signal by TCDD during the early stages of follicular development in the current model is unknown. In the first experiment of the current study, we test the hypothesis that the premature release of FSH and LH seen following exposure to TCDD is due to antagonism of estrogen action.
Ovulation in the rat is the result of the neural signal for ovulation transmitted to the GnRH neurons each afternoon [10, 11]. This neural signal is blocked by barbiturate anesthesia [12] and is thought to be transduced via ligand-independent activation of the progesterone (P4) receptor (PR) that in turn is upregulated by E2 as proestrus approaches [11]. We tested the hypotheses that 1) TCDD evokes a premature release of FSH and LH in the immature gonadotropin rat model through a similar pentobarbital-sensitive mechanism, and that 2) this mechanism requires activation of the progesterone signal similar to the endogenous neural signal for ovulation.
Data thus far largely support a hypothalamic action of TCDD during acute gonadotropin disruption in the female rat, although a pituitary action has not been definitively excluded [4]. Within the hypothalamus, dioxins may act on the GnRH neuron directly or target adjacent cell types (e.g., GABA neurons, astroglia). In the final experiment of this study, an immortalized GnRH neuronal cell line is used to test the hypothesis that TCDD acts directly on GnRH synthesis and secretion. Overall, this work presents experiments examining the direct impact of TCDD on a GnRH neuronal cell line as well as the interaction of TCDD with gonadotropin secretion in its requirements for estrogen, the daily neural signal for gonadotropin release and its putative mediator, the progesterone receptor, during synchronized follicular development in the rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature gonadotropin-primed rat model These experiments utilize the gonadotropin-primed immature rat model [2–4] to investigate the mechanism of acute TCDD action on gonadotropin secretion. In this model, induced follicular development is accompanied by rising estrogen, which elicits a preovulatory gonadotropin surge at approximately 56–58 h post-eCG followed by ovulation at approximately midnight on the third day (63 h post-eCG, although the current study focuses only on the early period of induced follicular development; Table 1). TCDD was donated by Dr. Karl Rozman (University of Kansas Medical Center). All chemicals were from Sigma (St. Louis, MO) unless otherwise described.
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The effects of exogenous E2 on the acute actions of TCDD on gonadotropin secretion Rats (n = 6 per treatment group) were pretreated with TCDD (32 µg/kg p.o., ED100 for ovulatory blockade) or corn oil vehicle on the morning of Day 25. This dose of TCDD has been shown to maximally stimulate a premature release of FSH and LH using the current model [4]. Endogenous E2 was supplemented with a long-acting E2 (estradiol cypionate [ECP], Upjohn, Kalamazoo, MI; 0, 0.01, 0.1, and 0.5 mg/kg s.c.) at the same time. Synchronous follicular development was induced with eCG (5 IU s.c.) at 0900 h on Day 26, and rats were killed 12 h later. TCDD has been shown to induce consistently elevated FSH and LH in this model by 12 h post-eCG in a dose-dependent fashion with an LOEL of 30 ng/kg TCDD p.o. [4]. Sera were collected and stored at -80°C until radioimmunoassay.
Effects of pentobarbital on the ability of TCDD to induce acute release of FSH and LH in the gonadotropin-primed immature rat model Immature female rats (n = 6 per treatment group) were pretreated with TCDD (0 or 32 µg/kg p.o.) and synchronous follicular development was induced with eCG as described above. Pentobarbital (32 mg/kg i.p.) or vehicle was administered on the morning of induced follicular development or on the following afternoon or evening (0, 6, or 9 h post-eCG). This dose of pentobarbital has been shown to block the normal preovulatory surge of FSH and LH in the rat by interrupting the daily neural signal for GnRH secretion [10, 12].
Effects of the progesterone and cortisol receptor antagonist, RU486, on the ability of TCDD to induce an acute increase in serum FSH and LH In order to test the role of the PR in the induction of premature FSH and LH by TCDD, immature female rats (n = 6 per treatment group) were treated with TCDD (8 µg/kg p.o., ED50 for ovulatory blockade) or corn oil vehicle, and follicular development was induced with eCG as described above. Additionally, rats received RU486 (0, 1, 10 mg/kg s.c.) 2 h prior to TCDD (0700 h, Day 25) and 24 h later. These doses are similar to those used previously to block the hypothalamic PR in the proestrous rat [13, 14]. A lower dose of TCDD was chosen for this experiment when initial studies indicated that RU486 facilitated, rather than blocked, premature gonadotropin release in response to TCDD. We reasoned that since our initial dose (32 µg TCDD/kg) maximally stimulated the early release of FSH and LH in response to TCDD [4], treatment effects further increasing gonadotropin concentrations could be difficult to detect. Rats were killed at 12 h post-eCG and sera collected as described above.
Radioimmunoassay LH, FSH, E2, and P4 were measured in duplicate serum samples (10–200 µl aliquots) by radioimmunoassay as described previously [15]. Interassay and intraassay coefficients of variation were less than 8%.
In Vitro Studies
Effects of TCDD on GT1–7 neuronal proliferation and GnRH accumulation Immortalized murine GnRH neurons (GT1–7 cells) [16] were obtained from Dr. Pamela L. Mellon (University of California, San Diego). Cells were grown (50 000/well, n = 4 per treatment/time group) to 75% confluence in DMEM (Dulbecco modified Eagle medium, Gibco, Gaithersburg, MD) supplemented with 10% fetal calf serum. Cells were washed and then treated with TCDD (0, 1, 10, and 100 nM) in serum-free DMEM. These doses of TCDD have been used previously to assess the impact of TCDD on ovarian and pituitary function in vitro [4, 17]. Medium was collected at 1, 2, 4, 8, 12, 24, and 48 h after treatment for GnRH measurement by previously validated radioimmunoassay [18]. Effects of TCDD on GT1–7 cell number were assessed following 48 h of treatment using the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) assay.
Effects of TCDD and E2 on GnRH promoter/reporter activity in transiently transfected GT1–7 cells GT1–7 cells [16] were grown (50 000/well) to 75% confluence in DMEM supplemented with 10% fetal calf serum. Cells were washed and then transfected with 0.1 µg pRSV-ß-galactosidase control vector and 0.5 µg of either a promoterless construct (pA3-luc), a constitutively expressed luciferase construct (pRSV-luciferase), or a luciferase reporter driven by 3.02 kb of the GnRH promoter (pA3–3026luc [19]). Transfection reactions were performed in serum free medium using the Lipofectamine Plus (Invitrogen, Carlsbad, CA) reagent and allowed to proceed for 3 h before the addition of culture medium and treatments. Treatments included TCDD (0, 1, or 100 nM) with E2 (0 or 10 nM). Transfected cells were cultured with treatments for 48 h, harvested, and assayed for ß-galactosidase and luciferase activity. GnRH promoter activity was expressed as fold change in luciferase activity corrected for ß-galactosidase expression.
Reverse transcription polymerase chain reaction (RT-PCR) for AhR Immortalized GnRH neurons (GT1–7 cells) and mouse ovarian surface epithelial cells (positive control [17]) were grown to approximately 75% confluence in 75 cm2 flasks. Total RNA was isolated from cultures using the TRIzol reagent (Invitrogen). Reverse transcription was performed using random primers with M-MLV (Maloney murine leukemia virus) RT at 42°C for 60 min followed by an increase to 94°C for 10 min. Previously validated primers [17, 20] for the rodent aryl hydrocarbon receptor and L19 housekeeping gene were used for RT-PCR within the same reaction tube (54° annealing T, 30 cycles). PCR products were visualized by electrophoresis on 2% agarose containing 1 µg ethidium bromide/ml.
Statistics
Values in figures are given as mean ± SEM. Data were analyzed by analysis of variance (ANOVA) with TCDD and treatment (i.e., ECP, RU486, or pentobarbital) as main effects. The interaction term TCDD x treatment was calculated as well. If a significant main effect or interaction was found, individual means were compared using Tukey test. P 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to determine the role of estrogen antagonism during acute gonadotropin disruption in female rats, exogenous E2 was administered to control and TCDD-treated animals prior to synchronous follicular development. Control animals injected with vehicle for TCDD had low concentrations (<1 ng/ml) of FSH and LH in the serum at 12 h following eCG administration, as was expected for this early stage of follicular development. In contrast to controls, TCDD markedly elevated serum gonadotropins as observed previously (Fig. 1, A and B). The long-acting E2, estradiol cypionate (ECP), interacted significantly with TCDD (TCDD x ECP, P < 0.05). This interaction is reflected in the significantly higher serum FSH and LH concentrations observed in rats receiving the highest dose of ECP with TCDD versus the lower gonadotropin concentrations seen in animals administered TCDD alone. Overall, E2 supplementation appears to have facilitated, rather than reduced, the acute release of FSH and LH following exposure to TCDD in the immature female rat.
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Serum E2 was elevated by ECP in a dose-dependent fashion (Fig. 1C). There was no effect of TCDD alone or in combination with ECP on the concentrations of 17ß-E2 in serum at 12 h post-eCG. ECP supplementation was associated with an elevation in serum P4 at the highest dose. TCDD had no significant effect on serum P4 in this model (Fig. 1D).
Effects of Pentobarbital and ECP on the Elevation of FSH and LH by TCDD
In order to assess whether the ability of TCDD to increase FSH and LH release acutely in the current model involves modulation of the neural signal for gonadotropin release or similar pathways, control and TCDD-treated rats were treated with pentobarbital (which blocks the endogenous neural signal for ovulation). TCDD alone increased serum FSH and LH (Fig. 2, A and B). Pentobarbital administered at 6 or 9 h (1500 or 1800 h) but not 0 h post-eCG (0900 h) decreased basal LH and FSH and ablated the ability of TCDD to elevate FSH and LH in vivo. ECP administration (0.5 mg/kg s.c.) did not interact significantly with the effects of TCDD or pentobarbital on serum gonadotropin concentrations.
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Serum E2 was elevated significantly by ECP, whereas TCDD failed to alter E2 concentrations in serum (Fig. 2C). Administration of pentobarbital was associated with significantly diminished E2 concentrations regardless of the presence of ECP. TCDD alone did not alter serum P4 concentrations at 12 h post-eCG (Fig. 2D). Pentobarbital alone diminished P4, particularly when given at 9 h post-eCG. The coadministration of ECP (0.5 mg/kg) ablated the effects of pentobarbital on serum P4 except when pentobarbital was administered at 9 h post-eCG. TCDD further diminished serum P4 when given in combination with pentobarbital (0 h post-eCG). However, TCDD moderated the decrease in P4 seen following administration of pentobarbital at 9 h post-eCG.
Effects of the Progesterone and Cortisol Receptor Antagonist Mifepristone (RU486) on the Ability of TCDD to Induce a Premature Gonadotropin Surge
In order to assess the role of the progesterone receptor in the mechanism of acute disruption of FSH and LH secretion during early follicular development in the female rat, the progesterone and cortisol receptor antagonist mifepristone (RU486) was used. TCDD (8 µg/kg p.o.) alone significantly increased LH and FSH concentrations at 12 h post-eCG (Fig. 3, A and B), but not so profoundly as seen with the higher dose of the dioxin (Fig. 1, A and B). RU486 failed to alter gonadotropin concentrations at either dose used. Administration of the higher dose of RU486 (10 mg/kg s.c.) was associated with a markedly greater elevation of FSH and LH in response to TCDD. This was reflected statistically as a significant (P < 0.05) interaction between the effects of TCDD and RU486.
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RU486 alone elevated serum E2 slightly but significantly at both doses (Fig. 3C). The highest concentrations of E2 were measured in rats receiving TCDD in combination with the RU486 (10 mg/kg). Basal concentrations of P4 at 12 h post-eCG were approximately 10 ng/ml serum. TCDD alone elevated serum P4 slightly, whereas RU486 alone markedly decreased serum concentrations of P4 at the highest dose. There was no significant interaction between the effects of RU486 and TCDD on P4 in the current model.
Effects of TCDD on GT1–7 Cell Numbers and GnRH Accumulation In Vitro
To test whether TCDD or E2 had direct effects on GnRH neuronal proliferation, GT1–7 cells received these treatments in vitro. Neither TCDD nor 17ß-E2 alone altered cell numbers in comparison with corresponding vehicle controls (data not shown). There was no significant interaction between the effects of TCDD and E2 on cell numbers as assessed by the MTT assay.
Treatment of GT1–7 cells with increasing concentrations of TCDD was associated with minimal effects on GnRH accumulation in conditioned media out to 72 h of culture (Fig. 4A). Cells cultured in the highest concentration of TCDD had slightly but not significantly elevated GnRH concentration following 6 h of culture, but this effect was not evident at lower doses or later time points.
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Similarly, TCDD failed to alter full-length GnRH promoter-reporter activity at doses of 1 or 100 nM in the presence or absence of E2 (Fig. 4B). The mRNA for the rat aryl hydrocarbon receptor was detected by RT-PCR in rat ovarian surface epithelium, but not in RNA isolated from immortalized GnRH neuronal cells (Fig. 4C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Administration of exogenous E2 during the later stages of synchronized follicular development restores a normal gonadotropin surge to eCG-primed immature rats treated with anovulatory doses of TCDD [9]. This same study demonstrated that TCDD markedly decreased the responsiveness of the hypothalamo-hypophyseal axis to the positive-feedback effects of E2 during the periovulatory period in the rat. The current study tested the hypothesis that the premature release of FSH and LH following exposure to TCDD in this model is due to the estrogen-modulatory actions of TCDD. Specifically, we hypothesized that the early release of gonadotropins following exposure to TCDD in the current model reflected a loss of E2-negative feedback. Estrogen supplementation sufficient to elevate serum E2 by over 50-fold failed to attenuate the aberrant premature release of FSH and LH in the current model (Fig. 1). Indeed, estrogen supplementation actually facilitated the release of gonadotropin in response to TCDD (Fig. 1). When combined with earlier studies, these results indicate that while a competitive blockade of the positive feedback effects of E2 might attenuate the preovulatory surge of FSH and LH following exposure to TCDD [9], a similar competitive antagonism of E2-negative feedback is not responsible for the effects of TCDD on gonadotropin release during early follicular development. Instead it appears that the acute stimulation of gonadotropin release by TCDD in immature female rats may involve an increase in the ability of E2 to elicit the release of GnRH from the hypothalamus.
The normal preovulatory gonadotropin surge in the rat is the result of a daily neural signal that is transduced to the GnRH neurons on proestrus [10, 11]. The GnRH, LH, and FSH surges resulting from this neural signal are blocked by the administration of barbiturates, including pentobarbital, on the afternoon of proestrus [12]. Previous work utilizing the immature gonadotropin-primed female rat model demonstrated that administration of pentobarbital between 1400 and 1800 h on Day 2 after eCG treatment inhibited the secretion of endogenous GnRH; ovulation was completely restored when exogenous GnRH was additionally administered with pentobarbital in this same animal model [21]. Pentobarbital hyperpolarizes postsynaptic membranes through GABAA receptor/Cl channel complexes [22–24]. In vitro, GABA receptor ligands have been shown to induce CYP1A1 [25], a member of the AhR gene battery, suggesting some commonality between AhR and GABA receptor pathways. Recently it was reported that during development in the rat, TCDD targets GABA-ergic neurons in the preoptic area of the brain [26]. The ability of pentobarbital to block LH and FSH release in response to TCDD in this model supports a hypothalamic, rather than hypophyseal, site of toxicant action, probably upon the hypothalamic GABA system. This is in agreement with previous studies [27].
The daily neural signal for ovulation in the rat is transduced via ligand-independent activation of the progesterone receptor on the afternoon of proestrus [10, 11, 28]. In contrast, progesterone (i.e., ligand-dependent PR activation) itself generally suppresses gonadotropin release when present during the estrous cycle and pregnancy. Estrogen-dependent upregulation of the hypothalamic PR during follicular development is required for the preovulatory gonadotropin surge, and administration of PR antagonists such as RU486 on the morning of proestrus blocks the endogenous LH surge [28]. Although there is an inhibitory effect of RU486 on the natural surge of LH and FSH at proestrus, the PR antagonist failed to interrupt the TCDD-induced premature gonadotropin surge in the current model. Indeed, antagonism of the PR actually increased the acute release of LH and FSH following exposure to TCDD. This may reflect blockade of a ligand-dependent (i.e., P4-mediated) action of the PR by RU486 that antagonizes the ability of TCDD to stimulate gonadotropin release, perhaps through modulation of the positive feedback of E2. Thus it appears that although the neural trigger for the natural FSH and LH surge and TCDD-induced gonadotropin release share a common sensitivity to barbiturates, the specific means through which these endogenous and exogenous agents exert their stimulatory actions on the GnRH system are distinct in the role of the PR.
TCDD did not alter GnRH accumulation and GnRH promoter-reporter activity in an immortalized murine GnRH cell line (GT1–7), suggesting that the dioxin acts only indirectly on the GnRH neurons in vivo. This cell line is commonly used as a model for the rodent GnRH neuron [16, 29, 30], as it allows study of purified and plentiful GnRH neurons that are currently impractical via primary culture approaches. In the current study, we failed to find expression of the Ah receptor in GT1–7 cells by RT-PCR. Previous studies have shown expression of the AhR in the hypothalamus in situ [31] and have suggested an action of TCDD on the hypothalamic GABA-ergic neuron [26]. In combination with our data, this further suggests that although TCDD does act at the level of the hypothalamus, the GnRH neuron is not a primary target of the toxicant.
An interesting aspect of TCDD action on gonadotropin release seen in this and previous studies (Fig. 1) [2, 4] is that the dioxin stimulates greater increases in FSH than LH in the immature female rat. In addition, Li et al. [4] observed significant increases in FSH at doses of TCDD approximately 100 times lower than the LOEL for LH secretion. Initially we hypothesized that this was due to effects of TCDD on inhibin, but preliminary studies failed to find any significant alterations in serum concentrations of the inhibin 
subunit in response to TCDD in the immature female rat [2]. An alternative explanation for variation between FSH and LH in the magnitude of response to TCDD is that TCDD may be altering the frequency of GnRH pulses in the hypothalamus. Such alterations in GnRH pulse frequency have been shown to change the relative amounts of FSH and LH released from the pituitary gland [32].
Overall, the results of this study establish an E2- and barbiturate-sensitive mechanism of endocrine disruption following exposure to TCDD in the immature gonadotropin-primed rat model. TCDD appears to stimulate gonadotropin release acutely through a mechanism above the level of the GnRH neuron that is antagonized by the progesterone receptor and facilitated by increases in E2. The hypothalamic GABA-ergic system appears to be a likely target of dioxin action during the acute disruption of gonadotropin secretion, although further study is needed to define this effect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Brian K. Petroff, Center for Reproductive Sciences, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. FAX: 913 588 7180; bpetroff{at}kumc.edu
Received: 18 August 2002.
First decision: 9 September 2002.
Accepted: 8 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-aminobutyric acid receptor binding and function as a mechanism of anesthesia. In: Roth RH, Miller KW (eds.), Molecular and Cellular Mechanisms of Anesthetics. New York: Plenum; 1986:165–177 This article has been cited by other articles:
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  Z. Shi, K. E. Valdez, A. Y. Ting, A. Franczak, S. L. Gum, and B. K. Petroff Ovarian Endocrine Disruption Underlies Premature Reproductive Senescence Following Environmentally Relevant Chronic Exposure to the Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Biol Reprod, February 1, 2007; 76(2): 198 - 202. [Abstract] [Full Text] [PDF]
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 S. A. Myllymaki, T. E. Haavisto, L. J. S. Brokken, M. Viluksela, J. Toppari, and J. Paranko In Utero and Lactational Exposure to TCDD; Steroidogenic Outcomes Differ in Male and Female Rat Pups Toxicol. Sci., December 1, 2005; 88(2): 534 - 544. [Abstract] [Full Text] [PDF]
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