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Department of Biological Sciences (I.H., H.O., R.J.H.), University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211; and Department of Obstetrics and Gynecology (R.G.R.), Rush Presbyterian St. Luke’s Medical Center, Chicago, Illinois 60612
Address all correspondence and requests for reprints to: Reinhold J. Hutz, Ph.D., Department of Biological Sciences, University of Wisconsin-Milwaukee, 3209 North Maryland Avenue, Milwaukee, Wisconsin 53211. E-mail: rjhutz{at}uwm.edu
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Introduction |
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The direct actions of TCDD on ovarian steroidogenesis are unknown, and the evidence is especially scarce, with respect to the human ovary. One study (10), involving in vitro administration of TCDD to human luteinized granulosa cells (LGC), showed a decrease in progesterone (P) production after a 24-h exposure. In another study (11), TCDD was shown to reduce E2 production by human LGC without an effect on P production. The discrepancy in the two studies may be caused by the high variability associated with human samples. Such variability is often a result of the ovarian stimulation protocol, the patient’s response to the stimulation, the age of the patient, and the cause of infertility. However, it seems that TCDD may exert some direct actions at the ovary to alter steroid secretion. We hypothesize that the effects of TCDD on female reproductive health occur through inhibition of ovarian steroidogenesis at one or more loci in the steroidogenic pathway. Additionally, because TCDD has previously been shown to manifest antiproliferative, antimitogenic, and apoptotic effects in nonovarian tissues (12, 13, 14), we suggest that its actions at the ovary may involve an increase in apoptotic cell death of the steroid-producing cells.
In the present study, we will determine whether TCDD is capable of mediating its effects at the human ovary, by inhibiting steroid secretion by LGC, and further evaluate whether the change in steroid secretion could be attributed to an induction in apoptotic cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human granulosa cell isolation and purification
Human LGC, used for the studies described herein, were recovered
from a total of six women participating in an in vitro
fertilization program and receiving treatments for ovarian stimulation
at Rush Presbyterian St. Luke’s Medical Center. Patient consent was
obtained before treatment. All women involved in this study were under
the age of 35 and had regular menstrual cycles, no male factor
infertility (had tubal infertility only), absence of endogenous LH
surge during stimulation, and an optimal serum E2 rise in
response to the stimulation protocol. Ovarian stimulation was
accomplished by pituitary desensitization and down-regulation using
leuprolide acetate (Lupron; TAP Pharmaceuticals, Abbott Park, IL) in
combination with human menotropins (human menopausal gonadotropin
and FSH: Pergonal and Metrodin; Serono Laboratories Inc.,
Randolph, MA). Follicular fluid aspirates were collected into 15-ml
centrifuge tubes approximately 36 h after a 10,000-IU human CG
(hCG) injection to simulate the midcycle LH surge. The LGC were
isolated by centrifugation at 300 x g for 5 min,
followed by an additional 5 min at 600 x g. This
resulted in a firm layer of LGC over a red blood cell pellet. Using a
Pasteur pipette, the layer of LGC was transferred into a fresh 15-ml
centrifuge tube (approximately 1 ml), and aggregates of LGC were
mechanically dispersed through a 1-ml pipette tip. Culture medium
[DMEM/F-12 (Gibco, Grand Island, NY) with 0.1% BSA] was added to the
cell suspension (up to 3 ml). The cell suspension was then layered over
50% Percoll (Sigma) and centrifuged at 300 x g for 30
min. The LGC were aspirated from the top of the Percoll solution and
pooled from all tubes into a fresh 15-ml tube and was diluted (up to 10
ml) with culture medium. The LGC were then washed twice by
centrifugation at 300 x g; the supernatant was
discarded each time, and the final vol brought up to 1 ml. The LGC were
separated from white blood cells using anti-CD45 magnetic immunobeads
(Immunotech, Westbrook, ME), as previously described by Best et
al. (15). The purified LGC were counted on a hemacytometer, and
the concentration was adjusted to 1 x 106 cells/ml.
Trypan blue exclusion dye was used to determine cell viability, which
was greater than 85%. The LGC were loaded onto 8-well Permanox-coated
Lab-Tek slides (Nunc, Naperville, IL) at 50,000 cells/well and
incubated at 37 C, with 5% CO2 in humidified air
overnight in 500 ml of culture medium (with 5% FBS). After this
initial incubation, the medium was aspirated and collected for steroid
hormone RIA analysis. Fresh medium, with or without TCDD (3.1
pM–3.1 mM) and with or without A4
(10 -7 M) or pregnenolone (10-7
M) was added to each of the wells, and the cells were
further incubated for an additional 48 h. The medium was collected
at 4, 8, 12, 24, 36, and 48 h after TCDD administration. Control
medium was comprised of 0.1% p-dioxane (identical to the
p-dioxane concentration in the highest TCDD concentration of
3.1 µM TCDD). Each treatment group was done in
triplicate, and each experiment was repeated at least three times and
comprised of cells obtained from one woman. Cells were obtained from
six different individuals. Cell counts of both TCDD-treated and control
samples were also evaluated at the end of each experiment. Both the
treatment and control groups similarly resulted in a less-than-25%
reduction in cell number; this reduction is caused by aspiration of
nonadhering dead cells.
Estrogen and P RIAs
E2 and P were measured in the medium at various time
intervals using the E2 and P Coat-A-Count kits (Diagnostic
Products Corp., Los Angeles, CA). All samples were assayed in duplicate
with the appropriate E2 or P standards. The intra- and
interassay coefficients of variation were derived from six replicate
aliquots of pooled human serum. The intraassay coefficients of
variation were 5.6% and 4.1% for E2 and P, respectively.
The interassay coefficients of variation were less than 10% for both
steroids.
Apoptosis determination
The APOPTAG In-Situ Apoptosis Detection Kit
(Fluorescein; Oncor, Inc., Gaithersburg, MD) was used to detect
apoptosis in cultured human LGC. The principles of the procedure
follow: Cells were fixed in 10% neutral-buffered formalin, washed, and
incubated with terminal deoxynucleotidyl transferase (TdT); negative
controls for nonspecific binding of the antibody were performed by
omission of TdT. The TdT enzyme catalyzes a template-independent
addition of deoxyribonucleotide triphosphate to the free 3'-OH ends of
double- or single-stranded DNA. In this case, TdT catalytically adds
residues of digoxigenin-nucleotide to the free 3'-OH ends that result
from DNA degradation, typical of early apoptotic events. An
antidigoxigenin antibody fragment is thus capable of attaching to the
digoxigenin nucleotides. The antibody fragment carries a fluorescein
molecule to the reaction site, which (upon excitation with light of 494
nm) generates a visible signal at 523 nm. Nonapoptotic cells were
observed with propidium iodide counterstain. The apoptosis experiments
were conducted on the cellular material used for the steroid
assays.
Transmission electron microscopy (TEM) of cultured human granulosa
cells
Human LGC were incubated on Permanox-coated Lab Tek slides
(Nunc) at 100,000 viable cells/well in 500 ml of media with or without
3.1 mM TCDD. Count and viability were determined using a
hemocytometer and trypan blue exclusion dye. Viability was greater than
85%. At the end of 24 and 48 h, the cells were washed in fresh
medium and fixed in 1.25% glutaraldehyde in 0.1 M
cacodylate buffer (CB), pH 7.4, for 30 min at room temperature. After
the initial fixation, the cells were scraped gently from the slides and
transferred to a 1.5-ml tube and pelleted by centrifugation for 5 min
at 500 x g. The cells were washed three times in CB.
After the final centrifugation, each cell pellet was resuspended in 50
ml of 2% sterile solution of sodium alginate (alginic acid-sodium
salt, low viscosity; Sigma) in saline and 50 ml of CB (0.2
M). The mixture was transferred to a syringe with a
25-gauge needle. Microspheres of the mixture were dropped into a 50-ml
sterile solution of 1% CaCl2 in saline. The microspheres
were washed several times in 0.1 M CB and postfixed in
1.5% OsO4 in 0.1 M CB, pH 7.3, for 1 h at
25 C. Three more washes in CB followed, and the specimens were taken
through a series of ethanol dehydrations: overnight at 50% and 4 C, 10
min at 75%, 10 min x 2 at 95%, 10 min at 100%, and 20 min at
100%. The dehydrated specimens were soaked in 25% Spurr’s resin in
100% ethanol for 1 h, in 50% resin for 1 h, and then in
75% resin overnight at 25 C. The final resin infiltration was
accomplished by embedding the specimens in a mixture of 100% Spurr’s
resin, which was allowed to polymerize overnight at 70 C. Semithin
sections were stained with 1% toluidine blue and viewed with a light
microscope to ensure the presence of cells in the preparation. Thin
sections were stained with methanoic uranyl acetate and then with lead
acetate before viewing in a Hitachi H600 TEM. Cells for this particular
experiment were pooled from two individuals.
Statistical analysis
The data for the E2 and P RIAs were
spline-transformed and analyzed using a one-way ANOVA with
Student-Newman-Keuls multiple comparison test. P <
0.05 was considered to be significant.
For apoptosis analysis, counts of apoptotic nuclei and/or apoptotic
bodies (green fluorescence) out of the total number of nuclei per field
were determined for each treatment group (control, 3.1 nM,
3.1 µM TCDD) at 24 and 48 h. Twenty fields were
counted for each sample, and a percent average apoptotic cell death was
calculated for each sample in triplicate. Percent apoptosis in specific
treatment groups was compared using 
-square goodness-of-fit with
Yates correction for continuity. Percentages were also partitioned
(subdivided) to determine significance among treatments.
P < 0.05 was considered to be significant.
For apoptosis analysis by TEM, counts of apoptotic cells out of the
total number of cells were determined for each treatment group (control
and 3.1 µM TCDD) at 24 and 48 h. Percent apoptosis
was determined by counting 200 cells in each of six grids from
individual blocks, ensuring that individual cells were not counted more
than once. Percent apoptosis in specific treatment groups was compared
using -square goodness-of-fit with Yates correction for continuity.
P < 0.05 was considered to be significant.
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Results |
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). At the pharmacologic concentration
(3.1 µM), TCDD affected E2 secretion in a
dichotomous fashion that was time dependent (Fig. 1). Specifically, we
observed a significant decrease in E2 secretion at 8, 12,
and 24 h, followed by a significant increase in secretion at 36
and 48 h. Analysis of total E2 accumulation in the
medium after 48 h showed that environmentally relevant doses, but
not the pharmacologic dose of TCDD, significantly inhibited
E2 secretion (data not shown). The addition of
A4 abolished the effect of TCDD on E2 (Fig. 2). In the presence or absence of
progestin substrate, P secretion was not affected by TCDD at any of the
time points evaluated (data not shown). TCDD treatment did not alter
total P accumulation after 48 h.
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)]. The addition of A4 to
the culture media did not change the observed increases in apoptosis at
24 or 48 h by the in situ technique. TEM showed
ultrastructural differences of LGC with 3.1 µM TCDD after
a 24- and 48-h incubation (Fig. 4). At
24 h, we observed increased nuclear chromatin condensation,
indicative of early apoptotic events, in cells treated with the 3.1
µM dose of TCDD, compared with controls (control =
15 ± 3.2%; 3.1 µM TCDD = 29 ± 2.5%);
at 48 h, the later stages of apoptosis, such as nuclear and plasma
membrane blebbing, were observed to be significantly greater with the
3.1 µM dose of TCDD, compared with controls (control
= 22 ± 4.5%; 3.1 µM TCDD = 59% ± 5.2%). In
addition to apoptotic effects (such as chromatin condensation, and
nuclear and plasma membrane blebbing), at 48 h the plasma membrane
in a portion of the TCDD-treated cells (30%) seemed to be ruptured;
and the presence of extracellular DNA, not membrane enclosed, was
evident (events that are not typical of apoptosis).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results indicated that in vitro administration of TCDD to human LGC was capable of altering E2 secretion into the culture medium. These effects, however, were not consistent, with respect to the dose of TCDD and the length of time in culture. Doses of TCDD that are considered to be environmentally relevant [3.1 pM, 1 part per trillion; 3.1 nM, 1 part per billion (16, 17)] resulted in decreased E2 secretion at 8, 12, and 24 h. We did not observe similar effects of TCDD at the later time points (36 and 48 h), suggesting that time in culture plays a central role in TCDD’s actions on human LGC. At the pharmacologic dose, TCDD exerted dichotomous effects on E2 secretion that were also time dependent; causing a significant reduction in E2 at 8, 12, and 24 h, followed by a significant increase at 36 and 48 h. Such time-dependent actions of TCDD have been observed previously in other tissues (13). Overall, the reduction in total E2 accumulation with the environmentally relevant dose of TCDD (3.1 nM) after 48 h in culture confirms previous results reported by others (11 21A ).
With the addition of A4 precursor to the media, we were able to circumvent the effects of TCDD on E2 secretion at all time points. This suggests that aromatase activity was not altered. Preliminary evidence from tritiated H2O assays for aromatase activity, in microsomal fractions of human LGC incubated with TCDD, support this idea (Dasmahapatra and Hutz, unpublished data). This evidence also concurs with results by Moran et al. (11) and further suggests that the effect of TCDD occurs early in the steroidogenic pathway, possibly by affecting CYP17 activity in androgen synthesis.
Although E2 secretion was significantly altered by TCDD treatment, our results indicate that TCDD did not affect P secretion by human LGC. This is consistent with data from a previous study by Moran et al. (11), in which no effects on P were observed with 10 nM TCDD, even after 8 days in culture and with the maintenance of a 2-IU/ml concentration of hCG. However, in an earlier study (10), TCDD was shown to decrease P production by human LGC after 24 h. The discrepancy between this and the present study may be because we did not maintain basal hCG levels in the medium. Furthermore, there is often great variability found when using human LGC from women undergoing ovarian stimulation for in vitro fertilization, and this may be the cause of the observed discrepancy between our study and that of Enan et al. (10) and between the two previous studies. The variability may be caused by the difference in stimulation protocol, the age of the woman, the cause of the infertility, and each woman’s individual response to the ovarian stimulation.
Because P was not affected, it is likely that the effect of TCDD on steroidogenesis does not involve cholesterol transport to the inner mitochondrial membrane (although steroidogenic acute regulatory protein was not evaluated), as was previously suggested for dioxin effects on Leydig cell steroidogenesis (22). The alteration of E2 secretion by TCDD is thus believed to involve intermediate steps after P synthesis and probably the provision of androgens for aromatization, or more specifically, cytochrome P450–17 Hydroxylase/17–20 lyase (P450c17) in the production of androgen precursors for estrogen synthesis. Of course, the repository for androgen in situ is the thecal cell. However, based on the fact that we were able to circumvent the effects of TCDD with the addition of A4 precursor, we suggest that TCDD depletes androgen precursor in the LGC themselves, so that it is not available for E2 synthesis. In the in vivo condition, this reduction in androgen bioavailability could be caused by an inhibition of CYP17 enzymes in the thecal cells. Currently, we have no data to support this contention, but future studies will entail coincubation of theca and LGC and thus allow us to better extrapolate TCDD’s actions to the in vivo state.
It is also possible that TCDD may alter estrogen metabolism in human LGC. TCDD has been shown previously to alter hydroxylase activity in the liver (23). Rather, we suggest that the decrease in E2 secretion observed with the environmentally relevant doses may actually be an increase in the conversion of estrogen precursors to the hydroxylated forms, such as the 2-OH, 4-OH, 6-OH, and 16-OH-estrone and E2 (catecholestrogens), which would not cross-react with our E2-specific antibody in the RIA. This hypothesis is based on earlier reports demonstrating hydroxylase activity, specifically 2-OH and 4-OH, in porcine ovarian granulosa cells (24) and the presence of hydroxylated estrogens, believed to be of follicular cell origin, in follicular fluid of a number of species, including humans (25). We are currently evaluating the potential role of TCDD in altering human LGC hydroxylase activity.
Other potential actions of TCDD on human LGC that could result in inhibition of steroid secretion might involve apoptosis induction. Previous studies have shown that apoptosis is a common mechanism by which atresia occurs in granulosa cells of ovarian follicles (26, 27, 28, 29). Moreover, TCDD, at environmentally relevant doses, has been shown to induce apoptosis, within hours, in tissues other than the ovary. Specifically, TCDD was shown to cause increased DNA fragmentation in thymocytes, before concentration-dependent increases in cytosolic Ca2+ level (12, 13, 30). In the present study, we observed an increase in apoptosis at 24 h with the pharmacologic dose of TCDD. Because this increase in apoptosis was only evident with the highest dose of TCDD, it does not explain the reduction in E2 that was observed at 8, 12, and 24 h with the lower doses of TCDD. It is possible, therefore, that the effects of TCDD on steroidogenesis may precede the apoptotic effects. Interestingly, by 48 h, we noted a dose-dependent increase in apoptosis. Ultrastructural analysis by TEM confirmed the in situ apoptosis occurs, with the pharmacologic dose of TCDD at both 24 and 48 h. At 24 h, we observed more cells, compared with control, exhibiting characteristics of early apoptotic events, such as chromatin condensation, specifically along the nuclear periphery. By 48 h, the pharmacologic dose of TCDD seemed to induce widespread plasma membrane and nuclear envelope blebbing, indicative of later stages of apoptosis. In addition to the increased apoptosis, we also visualized disruption of the cell membrane, such that the membrane seemed ruptured at one or more areas and cellular material extruded, suggesting that the high dose of TCDD caused changes in permeability. We therefore infer that the increased levels of E2 observed with TCDD treatment (3.1 µM) at the later time points may not necessarily be caused by an increase in E2 production, but rather by increased efflux of the steroid into the medium. This information, however, is still preliminary, and current studies are being conducted to investigate the increased permeability in human LGC by TCDD. These data, along with data from previous studies by McConkey et al. (12), strongly suggest that TCDD is capable of inducing apoptosis in human LGC but that other effects of TCDD on permeability, especially at high concentrations, may be occurring simultaneously.
Though the mechanism of TCDD-induced apoptosis is still unclear, evidence from studies in thymocytes suggests that it occurs via the AHR (12). We have previously shown the presence of the AHR in human LGC (8), suggesting that TCDD may mediate its effect on apoptosis through the induction of various apoptotic genes, after binding to the AHR. Studies using AHR blockers are currently being conducted to examine the potential involvement of the AHR in TCDD-induced apoptosis of human LGC.
The effects of TCDD on steroid secretion by human LGC may also be
receptor-mediated, especially because no effects were observed until
8 h in culture. However, in a preliminary study involving the
addition of 
-naphthoflavone (ANF; an AHR blocker) into the medium,
before and with the addition of TCDD, we were unable to block the
reduction in E2 secretion observed with the pM
and nM doses of TCDD. In fact, the addition of the AHR
blocker only enhanced the reduction in E2 (data not shown).
This result may have been caused by possible ß-naphthoflavone (an AHR
agonist) contamination in the high (1000-fold molar excess) dose of ANF
used, or by the fact that ANF itself sometimes exhibits weak AHR
agonist activity (31). The dose of ANF chosen was optimal for
competitively displacing TCDD at AHR sites, based on previous studies
(32).
In conclusion, we show, for the first time, that TCDD is capable of disrupting human ovarian steroid production, by acting directly at the follicular granulosa cells, to perturb E2 secretion and increase apoptosis, as observed by in situ immunofluorescence and by TEM, in a dose- and time- dependent fashion. We further suggest that TCDD, at least at pharmacologic doses, may exert effects on the cell membrane to alter permeability and, in this way, disrupt steroid secretion. We also show that the inhibition of E2 secretion by TCDD can be prevented by supplementation with androgen precursor. Collectively, these data suggest that TCDD alters steroidogenic function in human LGC by acting at one or more loci in the steroidogenic pathway (specifically, between the production of P and the provision of A4) and is capable of inducing apoptotic cell death by a mechanism(s) that is still unclear. Further studies are thus required to elucidate the exact locus/loci of TCDD action on ovarian steroidogenesis and the mechanisms involved in TCDD-induced apoptosis in the human ovary. The information gleaned from this and future studies is expected to enhance our understanding of the potential role of environmental pollutants in female reproductive health.
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Acknowledgments |
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Footnotes |
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2 Present address: Institute of Chemical Toxicology, Wayne State
University, 2727 Second Avenue, Detroit, Michigan 48201.
Received January 22, 1998.
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References |
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-naphthoflavone as an Ah receptor antagonist in
MCF-7 human breast cancer cells. Toxicol Appl Pharmacol 120:179–185[CrossRef][Medline]
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