TOXICOLOGICAL SCIENCES 77, 35– 40 (2004)
DOI: 10.1093/toxsci/kfg249
The Effects of Low-level Pb on Steroidogenic Acute Regulatory Protein
(StAR) in the Prepubertal Rat Ovary
Vinod Srivastava, Robert K. Dearth, Jill K. Hiney, Lisa M. Ramirez, Gerald R. Bratton, and W. Les Dees 1
Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843-4458
Received on July 15, 2003; accepted on September 12, 2003
protein, present in both rodent (Clark et al., 1995; Hilderbrand
1973; Sandhoff and McLean, 1996) and human ovaries
(Mushak, 1992), is the rate-limiting step essential in maintaining hormone-stimulated steroidogenesis (Chung et al., 1998;
Clark et al., 1995; Luo et al., 1998; Pescador et al., 1997;
Sandhoff and McLean, 1996). Specific effects of Pb on ovarian
function have been observed in rats and monkeys (Franks et
al., 1989, Hilderbrand et al., 1973; Stowe and Goyer, 1971).
Additionally, low Pb levels have been associated with decreased ovarian weight, fewer corpora lutea, and abnormal
estrous cycles in maternally exposed offspring (McGivern et
al., 1991). Recently, it has been shown that prepubertal females
exposed maternally to low levels of lead (Pb) exhibited suppressed circulating levels of both luteinizing hormone (LH)
and estradiol (E 2) (Dearth et al., 2002; Ronis et al., 1996,
1998). Because StAR gene expression is controlled by gonadotropins (Chung et al., 1998; Clark et al., 1995; Luo et al.,
1998; Pescador et al., 1997; Sandhoff and McLean, 1996), it is
expected that ovarian E 2 synthesis would be altered by decreased ovarian trophic support due to depressed LH. It is not
known, however, whether there is also an action of Pb directly
within the ovary. Therefore, the aim of the present study was
to determine if there were any intra-ovarian actions of Pb.
Estradiol (E 2) is suppressed in prepubertal females exposed
maternally to lead (Pb); thus, we assessed effects of Pb on ovarian
steroidogenic acute regulatory protein (StAR) as a potential mechanism for this action. Adult Fisher 344 females were dosed with 12
mg of lead acetate per ml of Pb acetate (PbAc) or sodium acetate
(NaAc; control), beginning 30 days prior to breeding and continuing until their pups were weaned. For the first part of this study,
animals from both groups were killed when 31 days old, at 0800 h,
for assessment of basal ovarian StAR gene expression. Results
indicated Pb decreased (p < 0.01) both StAR transcripts. In the
second part of the study, pregnant mare serum gonadotropin
(PMSG) was administered to half of the Pb-treated and control
animals at 0800 h. These animals, and animals from both groups
that did not receive PMSG, were killed and ovaries and blood
collected at 1600 h to assess ovarian StAR protein and E 2 responsiveness to gonadotropin stimulation. Pb decreased (p < 0.0001)
basal StAR protein expression and lowered (p < 0.001) E 2 levels in
animals that did not receive PMSG. PMSG induced (p < 0.0001)
StAR protein in both the Pb-treated and control animals, an action
associated with increased (p < 0.001) serum levels of E 2. These
results are the first to show that Pb alters basal StAR synthesis, but
does not alter gonadotropin-stimulated StAR synthesis, hence,
suggesting the primary action of Pb to suppress E 2 is through its
known action to suppress the serum levels of luteinizing hormone
and not due to decreased responsiveness of StAR synthesizing
machinery.
Key Words: lead (Pb) toxicity; steroidogenic acute regulatory
protein; pubertal development; Fisher 344 rats; luteinizing hormone; estradiol.
MATERIALS AND METHODS
Animals. As described previously (Dearth et al., 2002), adult Fisher 344
female rats purchased from Harlan Laboratories, Houston, TX, were housed
within the Laboratory Animal and Resources Facility at Texas A&M University and kept under controlled conditions of temperature (23°C), lights (on:
0600 h; off: 1800 h) and had ad libitum access to food (Harland Teklad Diet,
Madison, WI) and tap water.
The steroidogenic acute regulatory protein (StAR) is a 30kDa protein responsible for the transfer of cholesterol from the
outer to the inner mitochondrial membrane (Lin et al., 1995;
Orly and Stocco, 1999; Sugawara et al., 1995). Cholesterol is
then converted by the enzyme cytochrome P-450 side-chain
cleavage to pregnenolone (Luo et al., 1998; Miller, 1988;
Stocco and Clark, 1997) initiating a cascade of enzymatic
reactions resulting in the formation of E 2 (Miller, 1988). StAR
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. NaActreated 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.
1
To whom correspondence should be addressed. Fax: (979) 847-8981.
E-mail: [email protected].
Toxicological Sciences 77(1), © Society of Toxicology 2004; all rights reserved.
35
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SRIVASTAVA ET AL.
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 0C 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 E 2 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, HarborUCLA 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 0C until
assayed to determine the serum level of E 2. Ovaries were collected and frozen
at – 80 0C 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 manufacturer’s instructions (Tele-test, Friendswood, TX).
Northern-blot analysis was done as described previously (Srivastava et al.,
2001). Total RNA (30 ␮g) 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/cm 2) (Stratalinker
Crosslinker, Stratagene, La Jolla, CA). The membranes were prehybridized at
45°C for 4 h in a mixture containing 5⫻ SSC, 5⫻ Denhardt’s 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 ⫻ 10 6 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 1⫻ SSC/1% SDS and three times under high-stringency
conditions at room temperature (10 min) in 0.1⫻ 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 ␤-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 ⫻ g for 15 min at 4°C. The resultant
supernatant was centrifuged at 10, 000 ⫻ 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 ␮g) 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% SDS–
PAGE 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 (Westernblot Chemiluminescence Reagent Plus, NEN). The integrated optical density
of the bands was quantified using scanning densitometry.
Dosing solutions. A certified standard (Lot no. 8240 –11), 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 (E 2) 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.
RESULTS
At the times of breeding, through weaning on day 21, the
blood Pb (BPb) levels of the dams averaged 39.3 ⫾ 3.5 SEM
␮g/dl. This resulted in BPb levels averaging 2.9 ⫾ 0.28 SEM
␮g/dl and ovary Pb levels averaging 0.0161 ⫾ 0.005 SEM ppm
in their 31-day-old offspring (Fig. 1). The Pb levels in these
pups were similar to those known to cause delayed female
puberty (Dearth et al., 2002). Note, the Pb levels in both the
NaAc control dams and their offspring were all ⬍1.0 ␮g/dl.
Additionally, there were no differences in body weights, litter
sizes, fetal resorptions/litter, and pup deaths/litter between
control and Pb-exposed dams throughout the study.
The initial portion of this study was designed to determine
the effect of maternal Pb exposure on StAR gene expression in
prepubertal ovaries. Northern-blot analysis showed two major
StAR-specific transcripts at 3.8 and 1.7 kb in the ovary, as
EFFECTS OF LOW-LEVEL PB ON OVARIAN StAR
FIG. 1. Blood and ovary lead (Pb) levels from 31-day-old Pb-exposed
female rats: The bars represent the mean (⫾SEM) of n ⫽ 10 in each group.
depicted in Figure 2A by the composite autoradiogram. Figure
2B illustrates the densitometric values normalized with ␤-actin
mRNA by determining the ratio of the density of StAR mRNA
to that of ␤-actin mRNA. These data clearly show that mater-
37
nal Pb exposure resulted in a greater than 50% decrease (p ⬍
0.01) in both the 3.8 and 1.7 ovarian StAR transcripts, compared to the NaAc-exposed controls.
A second portion of this study was conducted to determine
the effect of maternal Pb exposure on prepubertal ovarian
StAR protein and E 2 responsiveness to gonadotropin stimulation. Figure 3A illustrates the representative Western-blot analysis of StAR protein, detected at 30 kDa, in ovaries from
Pb-treated and control animals with or without PMSG stimulation. Figure 3B is a composite of all animals, which depicts
the ability of Pb to decrease basal ovarian StAR protein expression by 27% (p ⬍ 0.0001) compared to NaAc-exposed
controls, a result that paralleled the decrease in mRNA expression shown in Figure 2. Importantly, PMSG stimulated a 216%
increase (p ⬍ 0.0001) in StAR protein expression in NaAc
control and a 400% increase (p ⬍ 0.0001) in Pb-treated ovaries
compared with the respective non-PMSG stimulated ovaries;
indicating that the StAR synthesizing machinery was functional in both groups. Furthermore, serum E 2 levels were
measured in these animals to confirm that the observed changes
in StAR protein expression were associated with changes in
steroid secretion. Figure 4 depicts a 46% decrease (p ⬍ 0.001)
FIG. 2. Effect of maternal Pb exposure on basal ovarian sodium acetate steroidogenic acute regulatory protein (StAR) gene expression in 31-day-old rats
by Northern-blot analysis: (A) Representative autoradiogram showing the expression of StAR mRNA in total ovarian RNA (30 ␮g) isolated from animals
exposed to NaAc (control, lanes 1–3), or lead acetate (PbAc) (lanes 4 – 6). The autoradiogram represents a 24-h exposure at –70°C. Note that the probe showed
two StAR-specific transcripts of 3.8 and 1.7 kb. The transcript for the internal control, ␤-actin, at 2.1 kb was detected by hybridization with a mouse ␤-actin
cDNA. ␤-Actin was exposed to autoradiographic film for 4 h at –70°C. (B) Composite drawings showing the densitometric quantitation of the bands from two
blots corresponding to the ovarian StAR transcripts. These data were normalized to the internal control ␤-actin mRNA; the densitometric units represent the
StAR/␤-actin (mRNA) ratios. Note the expression of mRNA was decreased (p ⬍ 0.01) for both StAR transcripts in animals that were exposed to Pb. Each bar
is the mean (⫾SEM) and depicts n of 6 lanes, with each lane representing the pair of ovaries from one animal; **p ⬍ 0.01 vs. NaAc control.
38
SRIVASTAVA ET AL.
FIG. 3. Effect of maternal Pb exposure on basal and PMSG-induced ovarian StAR protein expression in 31-day-old rats by Western-blot analysis: (A)
Representative Western immunoblot of StAR protein in ovaries from animals exposed to NaAc (control) offspring (lanes 1–3), PbAc (lanes 4 – 6), NaAc ⫹
stimulated with PMSG (lanes 7–9) or PbAc ⫹ stimulated with PMSG (lanes 10 –12). Total mitochondrial proteins (100 ␮g) were separated on 12% SDS–PAGE
and electroblotted onto a polyvinylidene difluoride membrane. The immunoblotting was performed with a rabbit polyclonal antibody. The bands representing
StAR protein were illustrated at 30 kDa. (B) Composite graph showing the mean (⫾SEM) densitometric quantitation of the bands from three blots corresponding
to the StAR protein. Note that basal StAR protein expression decreased (p ⬍ 0.0001) in offspring that were exposed to Pb. PMSG stimulation markedly increased
(p ⬍ 0.0001) StAR protein expression in both NaAc- and Pb-exposed offspring when compared to basal levels. Numbers within bars represents the number of
animals per group. Each lane represents a pair of ovaries from one rat; ****p ⬍ 0.0001 vs. NaAc control; ⫹ p ⬍ 0.0001 vs. Pb only.
in basal serum E 2 levels from 14.3 ⫾ 1.2 pg/ml in the controls
to 7.7 ⫾ 1.4 pg/ml in the Pb-exposed animals, coinciding with
the Pb-induced decreases observed in basal StAR gene and
protein expression shown in Figures 2 and 3, respectively. The
NaAc controls and Pb-treated animals that were administered
PMSG showed E 2 levels of 23.1 ⫾ 2.4 pg/ml and 24.9 ⫾ 1.8
pg/ml, respectively. These levels depict 61% (p ⬍ 0.001) and
223% (p ⬍ 0.0001) increases over the basal E 2 levels from the
control and Pb-treated rats that did not receive the PMSG
FIG. 4. Effects of maternal Pb exposure on basal and PMSG-induced
serum estradiol (E 2) in 31-day-old rats: Note that the pattern of serum E 2
changes paralleled those shown for ovarian StAR protein expression from the
rats depicted in Figure 4.3. Specifically, basal serum E 2 levels were suppressed
(p ⬍ 0.001) in offspring exposed to Pb compared to NaAc-exposed controls.
Pb did not block the PMSG-stimulated increase in serum E 2 (p ⬍ 0.001), a
response that was identical in the PMSG-stimulated, NaAc-exposed group.
Numbers within bars represents the number of animals per group; ***p ⬍
0.001 vs. NaAc; ⫹ p ⬍ 0.0001 vs. Pb only.
stimulation, indicating E 2 secretion was functional in both
groups.
DISCUSSION
As puberty approaches, the ovary is responsible for releasing
an increasing amount of E 2 necessary for the maturation of the
hypothalamic LHRH neuronal circuitry responsible for driving
the pubertal process (Ojeda et al., 1996a,b). The release of
LHRH/LH subsequently stimulates ovarian steroid production
(Ojeda et al., 1996a). Previous studies have shown that offspring exposed to Pb have suppressed levels of LH and E 2
(Dearth et al., 2002; McGivern et al., 1991; Ronis et al., 1998).
However, less is known regarding the effect of Pb on the
developing ovary; specifically, whether the metal can act directly within the gland to alter steroid production. It has become clear that StAR, the 30-kDa mitochondrial protein, is the
rate-limiting step in gonadotropin-stimulated steroid production (Luo et al., 1998; Stocco and Clark, 1997). It is well
known that in response to hormone stimulation, the StAR
37-kDa precursor protein is synthesized in the cytosol and is
directed to the mitochondria. This is where cleavage of the
mitochondrial targeting sequence occurs, yielding first to a
32-kDa form and then to the mature 30-kDa StAR protein
(Epstein and Orme-Johnson, 1991; King et al., 1995; Stocco
and Sodeman, 1991). At some point during the processing of
these proteins, the facilitation of cholesterol transfer occurs
from the outer to the inner mitochondrial membrane where the
39
EFFECTS OF LOW-LEVEL PB ON OVARIAN StAR
enzyme cytochrome P450scc resides (Epstein and Orme-Johnson, 1991; Stocco and Sodeman 1991). These earlier studies
have suggested that the active form of StAR is the 37-kDa
precursor form that facilitates the import of cholesterol into the
mitochondria. The definite mechanism of the regulation of
steroidogenesis by the different forms of StAR protein is not
yet fully known, but several recent studies have shown a direct
correlation between the synthesis of steroids and the synthesis
of the 30-kDa StAR protein in steroidogenic cells and thus,
represents an essential component in steroidogenesis (Huang
et al., 1997; Lin et al., 1995; Luo et al., 1998; Orly and Stocco,
1999; Stocco and Clark, 1997). Cholesterol is then cleaved to
pregnenolone to begin a cascade of enzymatic reactions resulting in the formation of E 2 (Miller, 1988). Recent in vitro
studies using mice showed that acute administration of Pb to
cultured Leydig cells caused a decrease in StAR protein expression and inhibited hCG-stimulated progesterone release
(Huang et al., 2002, 1997), suggesting Pb may act directly to
disrupt the StAR-synthesizing machinery necessary for ovarian
steroidogenesis.
The present study is the first to show altered intraovarian
StAR gene and protein expression and a subsequent decrease in
E 2 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 E 2 levels. As shown, both BPb and ovary Pb levels in
these females were very low, having almost returned to preexposure 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 E 2 (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 E 2 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 E 2 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 E 2 levels were
present in Pb exposed females; however, these E 2 levels were
significantly decreased compared to controls. Thus, because
LH facilitates the gradual increase in serum E 2 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 E 2 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 E 2 production. Low ovarian
Pb levels and the ability of PMSG to stimulate StAR protein
expression and subsequent E 2 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 E 2 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 E 2 synthesis.
ACKNOWLEDGMENTS
This work was supported by NIH Grants ESO9627 and ESO9106. The
authors wish to thank Dr. Douglas Stocco, Texas Tech University for the gift
of a rabbit antimouse polyclonal antibody to the StAR protein.
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