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Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, 84 Lipman Drive, New Brunswick, New Jersey 08901-8525, USA
(Requests for offprints should be addressed to M Uzumcu; Email: uzumcu{at}aesop.rutgers.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Early follicular development is primarily controlled by paracrine growth factors. Some growth factors, such as kit ligand and growth differentiation factor-9 stimulate follicular development, and their dysfunction or absence results in deficiencies in this process (Huang et al. 1993, Bedell et al. 1995, Dong et al. 1996). Other growth factors, including anti-Mullerian hormone (AMH), inhibit early folliculogenesis. In general, AMH is not expressed in primordial follicles, but only expressed in the primary follicle through small antral follicle stages. In pre-ovulatory, large antral follicles, AMH expression disappears or is very limited. AMH-expressing follicles appear to inhibit initial recruitment of primordial follicles, since increased initial follicular recruitment occurs in AMH-deficient mice (Durlinger et al. 1999). In addition, cultured neonatal mice ovaries treated with AMH have fewer growing follicles as compared with untreated ovaries (Durlinger et al. 2002). Furthermore, AMH may also inhibit gonadotropin-stimulated cyclic follicle recruitment, since AMH inhibits follicle-stimulating hormone (FSH)-stimulated aromatase activity and luteinizing hormone (LH) receptor expression in granulosa cells (di Clemente et al. 1994), thereby attenuating the effects of FSH on follicular development in the ovary. Thus, AMH appears to be a physiological brake in the ovary.
Neonatal estrogen treatment inhibits folliculogenesis (Ikeda et al. 2001) and stimulates AMH expression in the ovary (Ikeda et al. 2002). A recent study showed that estradiol treatment inhibits the primordial-to-primary follicle transition in neonatal ovaries in vitro, suggesting a direct action of the steroid on the ovary (Kezele & Skinner 2003). Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is an organochlorine pesticide used as a replacement for DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. MXC is estrogenic in vivo and has low affinity for the estrogen receptor (Hall et al. 1997). One of the primary metabolites of MXC is 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). HPTE shows estrogenic, anti-estrogenic, or anti-androgenic activities depending on the receptor subtype (Maness et al. 1998, Gaido et al. 2000). MXC is considered to be a model endocrine disruptor (Cummings 1997) with direct effects on the antral follicle (Miller et al. 2005).
Studies have shown the effect of relatively long-term MXC exposure on adult ovarian morphology and female fertility. Exposure to MXC during the first 14 days of life leads to irregular estrous cycles and causes the formation of either follicular cysts or ovarian atrophy in adult mice depending on the dose (Eroschenko et al. 1995). Rats were exposed to 5–150 mg/kg per day MXC 1 week before and 6 weeks after birth, and reproductive parameters were examined (Chapin et al. 1997). Doses of 50 and 100 mg/kg per day MXC stimulated early vaginal opening, inhibited ovarian development, caused irregular reproductive cycles, and reduced fecundity in rats. Exposure to 32 mg/kg MXC for 20 days reduces antral follicle number by atresia in mice (Borgeest et al. 2002). However, the effect of brief neonatal (P3–P10) exposure to MXC on pre-pubertal (P20) ovarian folliculogenesis and AMH production in the ovary has not been studied.
The objective of this study was to examine the effect of the estrogenic endocrine disruptor MXC on the rat ovary when administered during the primordial-to-primary follicle transition (P3–P10), which Kezele & Skinner (2003) demonstrated to be inhibited by estradiol-17ß. Tissues were collected for analysis at a pre-pubertal age (P20) before cyclic gonadotropin secretion starts in order to assess the effects of MXC on early folliculogenesis and AMH expression in the ovary. In the present study, as an exposure period P3–P10 was selected, since the formation of primordial follicles is mostly completed around P3 or P4 (Kezele & Skinner 2003). While most primordial follicles remain quiescent, a relatively large number of newly formed primordial follicles undergo the primordial-to-primary follicle transition right after follicle formation (McGee & Hsueh 2000). After the transition of primordial follicles into the primary follicle stage, they are destined for either ovulation or atresia (Hirshfield 1991). Both of these processes deplete the limited number of follicles, ending the reproductive lifespan of a female.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neonatal female Sprague–Dawley rats were injected daily (s.c.) with 1, 10, 50, 100, or 500 mg/kg per day methoxychlor between P3 and P10 (P0 = day of birth). Control rats were injected with vehicle, 25 µl DMSO:sesame oil (1:2). The s.c. injection route was used for a more precise dosing.
To facilitate reading, the 1 mg/kg per day MXC group will be referred to as ‘1MXC’, the 10 mg/kg per day MXC group will be referred to as ‘10MXC’, and so forth (Chapin et al. 1997). The MXC dose was calculated according to the body weight (BW) on the first day of the injections. The average BWs were not different between the groups and ranged from 9.4 to 9.6 g on P3. The doses that were used in this study are comparable with the doses that have been used in previous studies (5–800 mg/kg per day; Chapin et al. 1997, Hall et al. 1997). Environmental levels of MXC range from 40 to 160 ppm (mg/l) in waters downstream of MXC-sprayed areas (cited in Miller et al. 2005), which is within the range of doses in the present study. Since the actual MXC intake by humans averages only 100 ng/day (Gunderson 1988), MXC is not a specific concern for human health. However, many endocrine disruptors show estrogenic, anti-estrogenic, or anti-androgenic properties. Since MXC’s major metabolite, HPTE, has estrogenic, anti-estrogenic, and anti-androgenic actions, MXC was selected as a prototype endocrine disruptor in this study.
The animal care followed the Rutgers University Animal Care and Facilities Committee’s guidelines. On P20, the rats were euthanized by CO2 inhalation and the uteri and ovaries were collected. After organ weights were determined, one ovary was used for histology, follicle counting, and AMH immunohistochemistry (IHC) while the other was used for western blot analysis.
Ovarian histology
Ovaries were fixed in Bouin’s fixative for 2 h at room temperature and stored in 70% ethanol at 4 °C until embedding in paraffin. Paraffin-embedded tissues were sectioned at 4–5 µM. One of the sections from the largest cross section at the center of the ovary was used for hematoxylin and eosin (H and E) staining and was imaged using light microscopy to determine size of the ovary and follicle numbers and stages in the ovary.
Follicle numbers and stages
Using two sections from the largest cross section of the ovary, the number of follicles in each developmental stage was counted (Parrott & Skinner 1999). The follicles was classified according to previously published studies (Oktay et al. 1995). Primordial: an oocyte surrounded by squamous pre-granulosa cells of which not more than one is an enlarged granulosa cell. Primary: a relatively larger oocyte surrounded by at least two or more cuboidal granulosa cells. At the early primary follicle stage, some of the granulosa cells may still be squamous. Secondary (transitional): an oocyte surrounded by two to three layers of cuboidal granulosa cells. Pre-antral: an oocyte surrounded by more than three layers of granulosa cells with no apparent antrum. Antral: an oocyte surrounded by multiple layers of granulosa cells with an antrum. Unknown follicles: granulosa cells surrounded by a theca layer without a clear oocyte present.
AMH immunohistochemistry
One of the sections was used for IHC as previously described (Uzumcu et al. 2000). The sections were deparaffinized, rehydrated, and microwaved in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. The sections were then blocked with 1.5% normal serum for 10 min at room temperature. A polyclonal goat anti-AMH antibody (sc-6886, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the primary antibody. The sections were incubated with the primary antibody (1:200) overnight at room temperature. The negative control sections, instead of the primary antibody, were incubated with normal goat IgG or with primary antibody plus excess (10x) blocking peptide. Immunoreactivities were visualized using the ABC Staining System (Santa Cruz Biotechnology) following the manufacturer’s instructions. Sections were counterstained with Mayer’s hematoxylin.
Granulosa cell culture
The granulosa cells were prepared as described previously with some modification (Uzumcu & Lin 1994, Zachow & Uzumcu 2006). Briefly, to increase cell harvest, immature female rats (21–23 days old) were injected (s.c.) with 250 µg diethylstilbestrol (DES) in 100 µl sesame oil daily for 5 days. Eight hours after the last DES injection, the rats were killed. Ovaries were removed from the animal, isolated from the associated fat, oviduct, and bursa ovary, and then placed in ice-cold Dulbecco’s modified Eagle’s medium: Ham’s nutrient mixture F-12 (DMEM/F-12). Granulosa cells were isolated using a non-enzymatic needle puncture method with a sterile bundle of beading needles. Following two washes, the cells were plated at a density of approximately 4x105 viable cells/ml in a culture containing 10 ml DMEM/F-12 medium supplemented with 5% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin sulfate for 24 h. The cells were treated with HPTE (0.5, 1, 5, 10, or 25 µM) in 5 ml serum-free DMEM/F-12 for 24 h. The cells were harvested for AMH western blot analysis at the end of treatment. The experiment was repeated with granulosa cells harvested from immature rats that were injected (s.c.) with 15 IU equine chorionic gonadotropin (eCG).
Western blot analysis
Western blot analysis was performed as previously described (Uzumcu et al. 2002). The ovaries were lysed with 1x cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). The protein concentration in the supernatants was estimated using the DC Protein Assay Kit II (Bio-Rad). Approximately, 60 µg protein in the sample-loading buffer were boiled for 5 min and electrophoresed on a 10–12% SDS gel. The protein was subsequently transferred onto a nitrocellulose membrane and probed with specific antibodies for AMH and actin (AMH, sc-6886; actin, sc-1616; Santa Cruz Biotechnology). Following incubation with a secondary antibody, the specific antigen–antibody complex was visualized using ECL Advance Western Blotting Detection Kit (Amersham Biosciences) and viewed by autoradiography. The relative intensity was determined using actin as the loading control.
Statistical analysis
Organ weight, follicle numbers, and western blot data were analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The values were expressed as the mean ± S.E.M. Statistical analysis was performed and the difference between the means of treatments and the respective controls was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. For the ovarian weight, histology, IHC, follicle number, and whole-ovary AMH western blot analysis, experiments were repeated two to five times, using at least two to three animals for each treatment in each experimental repeat. Therefore, 5–11 animals were used for each treatment group. The granulosa cell culture AMH western blot analysis experiment was repeated thrice using one replicate (i.e. one culture flask) for each treatment in one experiment. For each experiment, the cells were obtained from seven to eight animals and combined. A statistically significant difference was confirmed at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Daily MXC treatment between P3 and P10 decreased ovarian weight and increased uterine weight by P20 (Fig. 1
). The decrease in ovarian weight was significant for 50, 100, and 500MXC (P < 0.01). Only 100 and 500MXC caused a significant increase in uterine weight (P < 0.01). None of the MXC doses caused systemic toxicological effects as indicated by general appearance, and no significant change in body weight occurred. On P20, the average BWs for all the groups ranged between 51.0 and 52.5 g (P > 0.05). In a separate experiment, the body weight of rats that were treated daily with MXC (1–500 mg/kg per day) did not significantly change during the treatments (data not shown).
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Daily MXC treatment between P3 and P10 caused a reduction in the size of the ovary and inhibited folliculogenesis (Fig. 2). The 50 and 500MXC treatments reduced ovarian size by approximately 35 and 80% respectively (Fig. 2C and E), as compared with the controls (Fig. 2A). The size reduction by 100MXC was approximately 75% (not shown). While control ovaries had many small, medium, and large antral follicles (Fig. 2B), there was a dose-dependent reduction in the number of antral follicles in the ovaries of 50MXC (Fig. 2D), 100MXC (not shown), and 500MXC (Fig. 2F). The size, histology, and follicle composition of 1 and 10MXC were similar to those of the controls (not shown). The 50MXC dose eliminated most of the large antral follicles, but some small and medium-sized antral follicles remained (Fig. 2D). Ovaries of 500MXC had few or no antral follicles (Fig. 2F). The 100MXC effect was similar to that of 500MXC (data not shown).
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In order to determine more precisely any effects of different doses of MXC on follicle numbers and stages, follicles in all stages of development were counted in two serial sections from the largest cross-sectional area at the center of the ovary (Fig. 3). On average, a section in control ovaries had 99.28 ± 8.0 total follicles that were distributed among primordial follicles, 9.0 ± 2.0; primary follicles, 22.9 ± 4.2; transitional (secondary) follicles, 4.1 ± 1.9; pre-antral follicles, 7.7 ± 3.3; and antral follicles, 12.3 ± 1.5. The total number offollicles was unaffected by any of the treatments. Both 100 and 500MXC reduced the antral follicle number significantly with a concomitant increase in the pre-antral follicle stage (P < 0.05). The number of follicles in primordial, primary, and transitional stages did not differ among all treatment groups (P > 0.05), nor did the number of unknown follicles differ between treatment groups (P > 0.05).
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Immunohistochemistry showed that AMH was produced by granulosa cells of primary, secondary, pre-antral, and small antral follicles (Fig. 4). In medium and large antral follicles, AMH immunostaining was reduced. This limited AMH production was primarily observed in cumulus granulosa cells (Fig. 4A; arrowheads). MXC-treated ovaries had many more AMH-positive follicles with stronger AMH immunostaining (Fig. 4C, E and G) as compared with the control ovaries (Fig. 4A). In 1 and 10MXC ovaries, the number of AMH-positive follicles and the intensity of the staining was similar to the control (not shown). In ovaries of 50MXC, the number of AMH-positive follicles increased and most follicles showed a more widespread and stronger staining (Fig. 4C and D). The majority of growing follicles, with the exception of some antral follicles (Fig. 4C; arrowhead), had strong, widespread staining in the granulosa cells. Antral follicles stained only in cumulus granulosa cells. In 500MXC ovaries, nearly all follicles other than primordial follicles showed strong AMH immunostaining (Fig. 4G and H). In 100MXC ovaries, the number of AMH-positive follicles and staining intensity was intermediary to the 50 and 500MXC (Fig. 4E and F). Higher magnification micrographs revealed stronger immunoreactivity in granulosa cells in 50, 100, and 500MXC ovaries (Fig. 4D, F, and H insets) as compared with granulosa cells in control ovaries (Fig. 4B inset). In control ovaries, granulosa cells had relatively limited AMH immunoreactivity in follicles beyond the primary follicle stage as compared with MXC-treated ovaries. Negative-control ovaries showed no specific immunoreactivity (data not shown).
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To quantify ovarian AMH production, western blot analysis was performed. The results of western blot analysis supported our observations in IHC and showed that ovaries of 50, 100, and 500MXC-treated females contained 1.6 ± 0.2, 1.85 ± 0.6, and 2.2 ± 0.5 times more AMH protein than the control ovaries respectively (Fig. 5A and B), the differences being statistically significant (P < 0.05). The AMH protein production in ovaries of 1 and 10MXC was not significantly different from control ovaries (P > 0.05). The molecular weight of the AMH band was approximately 65 kDa, which is similar to a previously reported result for western blot analysis of bovine AMH separated under reducing conditions (Wilson et al. 1993).
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To assess whether increased ovarian AMH production following MXC treatment in vivo was due to a direct action of MXC or its metabolites in the ovary, we examined the effect of HPTE on granulosa cells as HPTE is more active (e.g. has a higher affinity for estrogen receptor (ER)) than MXC. While 0.5 µM HPTE stimulated AMH production, this was not significant (P > 0.05). However, 1 and 5 µM HPTE significantly stimulated AMH production (P < 0.05; Fig. 6). Higher doses of HPTE (10 and 25 µM) stimulated the AMH production but this was not significant (P > 0.05), and 25 µM HPTE appeared to be toxic to the cells since some of the cells detached (not shown). A similar result was obtained when the experiment was done using granulosa cells harvested from immature rats, primed with 15 IU eCG (s.c.) for 24 h (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MXC treatment causes a reduction in the weight and size of the ovary. In addition, while the ovaries of each MXC treatment had approximately the same total number of follicles, MXC-treated ovaries had a dose-dependent reduction in the number of antral follicles with a concomitant increase in pre-antral stage follicles. This is similar to previous observations that neonatal estrogen treatment causes a reduction in the number of medium and large antral follicles (Ikeda et al. 2001, 2002). A recent in vitro study has shown that estrogen treatment reduces the number of growing pre-antral follicles, suggesting that estrogen inhibits the transition from primordial to primary follicle stage (Kezele & Skinner 2003). In the present study, MXC-treated ovaries had fewer antral follicles without an apparent reduction in total follicle number. This suggests that the effect of MXC on follicular development can be at any stage, such as the primordial-to-primary follicle transition, growth of the primary follicle into a more advanced pre-antral follicle, or recruitment of pre-antral follicles into the antral follicle stage. Based on the follicle numbers, it seems that the inhibition of MXC may be on the latter: the recruitment of pre-antral follicles into the antral follicle stage. Further investigation, such as follicle counts in multiple sections throughout the entire ovary, is needed to confirm this finding.
Due to its estrogenic actions, MXC may provide a negative feedback on the hypothalamus and pituitary and reduce gonadotropin secretion, which can inhibit ovarian folliculo-genesis in treated rats. The pituitary hormones are present in pre-pubertal rats. The level of FSH is low at birth and reaches its peak value between P12 and P15, after which it returns to the initial low level and stays low until P45 (Ojeda & Ramirez 1972). Estradiol normally cannot induce a negative feedback on the hypothalamus and pituitary in pre-pubertal rats because of serum 
-fetoprotein, which binds to serum estradiol and does not allow it to move into tissues, such as the hypothalamus and pituitary (Greenstein 1992). Whether MXC can transfer into these tissues is not known. However, if it does, MXC can potentially act on these tissues because ER and ERß are expressed in the hypothalamus and pituitary of neonatal female rats (Nishihara et al. 2000, Ikeda et al. 2003). It has been speculated that neonatal MXC exposure leading to cycle irregularities and ovarian pathology in mice is probably due to altered hypothalamic–pituitary function (Eroschenko et al. 1995). However, whether MXC affects the hypothalamus and pituitary is controversial. Injection of MXC (8–64 mg/kg per day) in adult mice for 20 days did not change the serum FSH levels or levels of the FSH receptor in the ovary (Borgeest et al. 2004). In addition, feeding MXC (24–1200 ppm) to pregnant rats between embryonic day 15 (E15) and P10 does not affect the size of the sexually dimorphic nucleus of the pre-optic area of the hypothalamus in the female offspring (Masutomi et al. 2003). On the other hand, perinatal (E15–P10) exposure to MXC has been reported to inhibit serum LH levels in adult female rats (Suzuki et al. 2004). Similarly, treatment with 50 or 100 mg/kg per day MXC for 1 week before and 6 weeks after birth suppressed FSH levels during estrus (Chapin et al. 1997). In addition, preliminary results from our laboratory suggest that MXC treatment between P3 and P10, suppressed serum FSH levels on P8–P16 (Clayton & M Uzumcu, unpublished observations). Studies are underway to confirm the preliminary results and to further investigate the exact role of suppressed gonadotropins in the observed inhibition of folliculogenesis in the present study.
In addition to its potential action through the hypothalamic-pituitary-ovarian (HPO) axis, MXC and/or its metabolites probably act directly on the ovary. It is possible that MXC stimulates ovarian AMH production directly, which in turn inhibits folliculogenesis. In the present study, the increased intensity of the AMH immunostaining in individual granulosa cells or follicles in MXC-treated ovaries suggests that MXC acts on the ovary directly. This suggestion was clearly supported by our results which show that HPTE, one of the major metabolites of MXC, significantly stimulated AMH production in DES-primed rat granulosa cells in vitro. This novel finding is somewhat expected, since MXC has been shown to directly increase antral follicle atresia through several mechanisms, including the Bcl-2 and Bax-mediated pathway (Miller et al. 2005), oxidative stress-mediated pathway (Gupta et al. 2006), and estrogen signaling-mediated pathway (Miller et al. 2006, Tomic et al. 2006) in a follicle culture system. In addition, estrogen and estrogenic compounds, such as MXC and its metabolites, are known to regulate ovarian AMH gene expression. The AMH promoter has a 13 base pair sequence that differs only by one nucleotide from the estrogen responsive element (ERE) of the vitellogenin gene found 1722 base pairs upstream of the human gene. Co-transfection of ER and ERE inverted repeat as 35 base pair oligonucleotide linked to a reported gene confers estrogen responsiveness on a heterologous reporter (Guerrier et al. 1990). Elevated AMH can inhibit folliculogenesis, since AMH inhibits the primordial-to-primary follicle transition (Durlinger et al. 2002) and FSH-induced follicular development (di Clemente et al. 1994) in the ovary. Data presented from the present study as well as studies by others suggest that the inhibition offolliculogenesis that was observed in this study can be due to direct action on the ovary –probably through increased ovarian AMH production.
In conclusion, this study shows that neonatal exposure to the endocrine disruptor MXC inhibits ovarian follicular development and reduces antral follicle numbers in rats. In addition, MXC, possibly through its major metabolite HPTE, stimulates AMH production in the ovary. Thus, the inhibitory effect of MXC on the ovary may, at least in part, be directly on the ovary and mediated by the local paracrine factor AMH. This study highlights the potential adverse effects of endocrine disruptors on ovarian pathology and female fertility, which can be mediated by local paracrine factors such as AMH.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Received in final form 31 August 2006
Accepted 8 September 2006
Made available online as an Accepted Preprint 27 September 2006
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P < 0.01.
