Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4458
Received July 15, 2003; accepted September 12, 2003
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ABSTRACT |
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Key Words: lead (Pb) toxicity; steroidogenic acute regulatory protein; pubertal development; Fisher 344 rats; luteinizing hormone; estradiol.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Experimental protocol.
Starting 30 days prior to breeding, animals were gavaged daily with a 1.0-ml solution of lead acetate (PbAc) containing 12 mg of Pb/ml until their pups were weaned 21 days after birth. Additional animals served as controls and received a daily 1.0 ml dose of an equimolar sodium acetate (NaAc) solution by gavage. Following the 30-day dosing period, both Pb-treated and control females were bred to non-Pb-exposed proven males and the morning that vaginal plugs were observed was considered day-one of gestation. Dams dosed with Pb were bled via tail tip on days 0, 15, and 30 prebreeding, day 10 of gestation, and days 1, 10, and 21 of lactation. NaAc-treated rats were bled on day 0 and at the end of the experiment, in order to verify that they were not exposed to Pb throughout the study. Rats were dosed at approximately 1:00 P.M. daily to help ensure uniform absorption.
All animals were weaned at 21 days, and females were housed, 4 per cage, until they were thirty days old. The following morning, both the Pb-exposed and NaAc-treated control animals were divided such that the study would have two parts. The first consisted of 31-day-old Pb-treated and NaAc-treated control animals that were killed by decapitation at 0800 h, and their ovaries, which were removed and frozen at -80°C for subsequent determination of basal StAR gene expression. Animals for the second part of the study were used for the determination of StAR protein and serum E2 levels, following gonadotropin stimulation. For this, both groups were subdivided such that half of the Pb-exposed animals and half of the NaAc control animals received subcutaneous injections of 15 IU of pregnant mare serum gonadotropin (PMSG; purchased from Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, Torrance, CA) at 0800 h. The other half of the animals from each group were not injected with PMSG. At 1600 h, all of the animals were killed by decapitation and trunk blood was collected. For each sample, an aliquot was removed for measurement of Pb concentrations, and the rest of the sample was allowed to clot, then was centrifuged and stored at -80°C until assayed to determine the serum level of E2. Ovaries were collected and frozen at -80°C for subsequent Western-blot analysis for StAR protein. Pb concentrations in both blood and ovary were determined by atomic absorption spectrophotometry.
Northern-blot analysis.
Total RNA was prepared from ovaries, using RNA Zol B followed by precipitation with isopropanol and ethanol washes, according to the manufacturers instructions (Tele-test, Friendswood, TX). Northern-blot analysis was done as described previously (Srivastava et al., 2001). Total RNA (30 µg) was denatured in the presence of 6% formaldehyde and 50% formamide at 650°C for 15 min, and run on 1% agarose gels containing 2.2 M formaldehyde. Following size fractionation, RNA was transferred onto a nylon membrane (NEN Life Sciences Products, Inc., Boston, MA), followed by UV cross-linking of RNA (0.12 J/cm2) (Stratalinker Crosslinker, Stratagene, La Jolla, CA). The membranes were prehybridized at 45°C for 4 h in a mixture containing 5x SSC, 5x Denhardts solution: 50% formamide, 20 mM sodium phosphate (pH 6.8), 2.5 mM EDTA, and 0.1 mg/ml denatured salmon testis DNA. Hybridization was carried out in the same solution at 45°C for 16 h with 10% dextran sulfate and 2 x 106 cpm StAR cDNA probe labeled with [
-32P] dCTP (3000 Ci/mmmol;NEN) by using Random Primers DNA-labeling System (Gibco-BRL, Rockville, MD). Unincorporated nucleotides were separated from radiolabeled DNA probes by using miniQuick spin oligo columns (Roche Molecular Biochemicals, Indianapolis, IN). After hybridization, the blots were washed three times at room temperature (10 min) in 1x SSC/1% SDS and three times under high-stringency conditions at room temperature (10 min) in 0.1x SSC/0.1%SDS. After washing, the membranes were exposed to XAR-5 film for 24 h with an intensifying screen at -70°C. Following, Northern-blot analysis with StAR cDNA, blots were stripped and rehybridized with a mouse ß-actin cDNA (Ambion, Inc., Austin, TX) labeled with Random Primers DNA-labeling kit (Gibco-BRL, Rockville, MD). The autoradiograms were quantified using scanning densitometry. The integrity of the RNA was confirmed by ethidium-bromide staining of the gel, which showed the intact ribosomal RNAs, i.e., 18S and 28S subunits, and whether equal amounts of RNA had been loaded in each lane. The RNA transcript size was determined by comparison with an RNA molecular weight (mw) marker in lanes adjacent to the sample RNA lanes.
Isolation of mitochondria.
Ovaries were homogenized in a sucrose buffer containing 10 mM Tris Cl, 0.25 M sucrose, and 0.1 mM EDTA, pH 7.4. The homogenates were centrifuged at 600 x g for 15 min at 4°C. The resultant supernatant was centrifuged at 10, 000 x g for 15 min at 4°C to pellet the mitochondria, which was then resuspended in sucrose buffer and an aliquot was removed for protein determination by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).
Western-blot analysis.
Western-blot analysis of mitochondrial protein was performed as described previously (Srivastava et al., 2001). Briefly, the mitochondrial proteins (100 µg) were solubilized in a sample buffer containing 25 mM Tris Cl, pH 6.8, 1% SDS, 5% mercaptoethanol, 1 mM EDTA, 4% glycerol, and 0.01% bromophenol blue, and were separated onto 12% SDSPAGE under reducing conditions, along with prestained mw markers (Amersham Pharmacia Biotech, Piscataway, NJ). The separated proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes using a mini trans-blot apparatus (Bio-Rad, Hercules, CA). The transfer was preformed at a constant voltage of 100V for 1.5 h in a buffer consisting of 48 mM Tris, 39 mM glycine, pH 8.3, 0.037% SDS, and 20% methanol. Following transfer, membranes were blocked overnight at 4°C in the presence of 5% nonfat dry milk in phosphate buffered saline (PBS) and subsequently incubated at room temperature for 3 h with a rabbit antimouse polyclonal antibody to the StAR protein (diluted 1: 1000 in PBS containing 0.01% T-20), which was kindly provided by Dr. Douglass Stocco, Texas Tech University, Lubbock, TX). After washing, the membranes were incubated with horseradish peroxidase-labeled donkey antirabbit IgG at a dilution of 1: 14,000 for 2 h at room temperature. After another wash, the immunoreactive 30 kDa StAR protein was detected by the enhanced chemiluminescence method (Western-blot Chemiluminescence Reagent Plus, NEN). The integrated optical density of the bands was quantified using scanning densitometry.
Dosing solutions.
A certified standard (Lot no. 824011), made with Pb acetate-trihydrate and containing 100 mg Pb/ml in 2% nitric acid, was purchased from Scientific Equipment Company, Aston, PA and served as the stock Pb solution. Using the method described previously (Dearth et al., 2002), a dosing solution of 12 mg/ml of Pb was made fresh daily, immediately before dosing, in a 50-ml centrifuge tube. The pH of the NaAc dose given to the control animals was adjusted to 5.5 with ultrapure nitric acid, to match that of the Pb-dosing solution.
Blood and tissue Pb measurements.
All Pb levels were measured by a Perkin Elmer SIMAA 6000 simultaneous multi-element atomic absorption spectrophotometer (AA) with a transversely heated graphite furnace, Zeeman background correction system, AS-72 autosampler, and an AA Winlab applications package (Perkin Elmer Corporation, Norwalk, CT). Blood and ovary Pb levels were processed and analyzed using methods described previously (Dearth et al., 2002).
Radioimmunoassay and statistical analysis.
Serum estradiol (E2) was measured by an RIA kit purchased from Diagnostic Products Corp. (Los Angeles, CA). The assay sensitivity was 8 pg/ml and inter- and intra-assay coefficients of variation were <10%.
Differences between NaAc- and Pb-treated animals were analyzed using an unpaired Student t-test, assuming random sampling, Gaussian distribution, and independent observations. Two-sided probability (p) values were used, and those less than 0.05 were considered to be significantly different. The IBM PC software programs, INSTAT and PRISM (GraphPad, San Diego, CA, USA), were used to calculate and graph the results.
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RESULTS |
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DISCUSSION |
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The present study is the first to show altered intraovarian StAR gene and protein expression and a subsequent decrease in E2 production by ovaries from late juvenile offspring that were maternally exposed to Pb at levels equal to those of human concern (CDC, 1997, 1991
). Specifically, 31-day-old offspring exposed to Pb throughout gestation and lactation showed a significant decrease in both basal StAR gene and protein expression. These results coincided directly with decreased serum E2 levels. As shown, both BPb and ovary Pb levels in these females were very low, having almost returned to pre-exposure levels; hence, suggesting an effect on the ovary that is long-lasting.
During the late juvenile phase of pubertal development, the release of LH from the pituitary plays an important role in ovarian maturation by increasing gonadotropin receptor numbers and facilitating production of E2 (Ojeda et al., 1986a). This trophic stimulation of receptors on the ovary causes, through several signaling pathways, activation of the StAR gene, thus producing an increase in StAR protein expression and, subsequently, ovarian steroids (Chung et al., 1998
; Clark et al., 1995
; Pescador et al., 1997
; Sandhoff and McLean, 1996
). Previous studies in prepubertal and adult rats showed that Pb decreased both LH and FSH testicular receptor concentrations (Kempinas et al., 1994
; Wiebe et al., 1982
). Additionally, Pb has been shown to alter ovarian LH receptor concentrations in prepubertal females (Wiebe et al., 1988
); however, it is important to note that the animals in those studies were exposed to Pb levels higher than those relative to human concerns. In the current study, prepubertal ovarian receptor responsiveness was not hindered by low-level Pb exposure, as shown by the ability of PMSG to stimulate StAR protein and E2 secretion. Importantly, the exposure levels of Pb in this study were markedly lower than those previously associated with altered LH receptor populations (Wiebe et al., 1988
). Although the present study did not assess the possibility that more subtle changes in LH receptor populations could have occurred with the low-level Pb exposure used, the responsiveness of the receptors to a dose of PMSG used routinely to assess ovarian function was not altered. The stimulatory response PMSG has on E2 release replicates the ability of both FSH and LH to induce ovarian steroidogenesis (Dees et al., 1990
; Mizutani et al., 1997
). Maternal Pb exposure, however, only alters LH, but not FSH secretion (Dearth et al., 2002
; unpublished observation). Apparently FSH had a limited ability to influence ovarian function, because basal E2 levels were present in Pb exposed females; however, these E2 levels were significantly decreased compared to controls. Thus, because LH facilitates the gradual increase in serum E2 necessary for a normal progression through puberty (Ojeda et al., 1996a,b), we suggest that Pb exposure does not affect the ovarian StAR synthesizing machinery, and that the Pb-induced decrease in basal E2 levels observed were likely due to the suppressed trophic support of LH to the prepubertal ovary. In support of this, we have observed previously that serum LH levels in 30-day-old Pb-exposed pups were 0.25 ng/ml, compared to 0.7 ng/ml from their age-matched controls (Dearth et al., 2002
).
In conclusion, these results showed a decrease in both basal ovarian StAR gene and protein expressions in late prepubertal female offspring from Pb-exposed dams. This Pb-induced effect coincided with a decrease in E2 production. Low ovarian Pb levels and the ability of PMSG to stimulate StAR protein expression and subsequent E2 release in Pb-exposed females strongly suggests that the metal does not directly affect ovarian responsiveness to gonadotropin stimulation. Since it is known that Pb causes a decrease in serum LH and E2 levels in maternally exposed female offspring (Dearth et al., 2002; Ronis et al., 1998
, 1996
), we suggest that Pb acts at the hypothalamic-pituitary level to disrupt the peripubertal gonadotrophic support contributed by LH, necessary to drive StAR production and subsequent E2 synthesis.
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ACKNOWLEDGMENTS |
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NOTES |
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