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Glutathione S-Transferase (GSTA1, GSTA2) Subunits Is Mainly Associated with Steroidogenically Active Cells and Regulated by Gonadotropins in Bovine Ovarian Follicles1
Centre de recherche en reproduction animale (F.R., S.B., J.S., D.W.S., J.G.L.), Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, J2S 7C6, Canada; and Faculté de médecine vétérinaire (J.-F.B.), Université de Liège, B4000 Sart Tilman, Liège, Belgium
Address all correspondence and requests for reprints to: Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, Québec, Canada, J2S 7C6. E-mail: lussij{at}medvet.umontreal.ca
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class glutathione S-transferase, named
bGSTA1 and bGSTA2. Both bGSTA1 (25.4 kDa, pI 8.9; 791 bp cDNA; GenBank
Accession No. BTU49179) and bGSTA2 (25.6 kDa, pI 7.2; 959 bp cDNA;
GenBank Accession No. AF027386) have 222 amino acids. The deduced amino
acid sequences were compared and showed 82% (bGSTA1) and 74% (bGSTA2)
identity to human GSTA1, whereas bGSTA1 and bGSTA2 are 81% identical
to each other. The bGSTA2 represents a novel GSTA subunit because it
harbors a specific 16 amino acid sequence not found in any other
species and GST classes. Northern blots showed that bGSTA1 and bGSTA2
are coexpressed and are tissue specific with single transcripts of 1.2
kb and 1.4 kb, respectively for bGSTA1 and bGSTA2. The messenger RNA
(mRNA) were detected in GC, corpus luteum, adrenal gland, testis,
liver, lung, thyroid, kidney and cotyledon, and the relative abundance
of their mRNA varied. Ratios of bGSTA1/bGSTA2 mRNA vary between
tisssues, indicating that expression of these genes is controlled
differently. Immunohistochemistry observations revealed that expression
of GSTA is cell specific, being associated with GC and theca cells,
small luteal cells, Leydig cells, hepatocytes, adrenal cortex, specific
chromaffin cells in the adrenal medulla, renal proximal convoluted
tubular cells, and Clara cells in the bronchioles. Studies in
vivo showed that levels of mRNA for bGSTA1 were elevated in
follicular wall of preovulatory follicles before hCG treatment, but
decreased by 77% 12 h after hCG injection. However, in FSH
stimulated preovulatory follicles, the decrease in mRNA for both GSTAs
was only 21% at 24 h following hCG injection. We concluded that
bGSTA1 and bGSTA2 expression is tissue- and cell-specific, is
associated with steroidogenically active cells, and is hormonally
regulated by gonadotropins in the bovine ovarian follicle. |
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In the present study, we have partially purified and raised polyclonal
antibodies against two proteins contributing to the 28 kDa granulosa
cell protein group that were initially named p28 and p29. We report
herein their characterization by complementary DNA (cDNA)
immunoscreening of a luteal cell expression library and reveal that
these cDNAs encode for two uncharacterized bovine glutathione
S-transferase subunits (GSTA). We named these subunits bGSTA1 and
bGSTA2, based on the proposed human nomenclature (5). The GSTs are a
multigene family of related proteins that have been divided into five
evolutionary classes of , µ, 
, 
, and 
on the basis of
their biochemical and immunological characteristics. Within each class,
GST isoenzymes were characterized and their number varies between
species (6, 7, 8). These five classes of GST were reported to be localized
in the cytoplasm, whereas the cell membrane associated GST, the
microsomial GST and the C4 leukotriene synthetase, which differs in
structure to the cytosolic GST, were classified differently. The
described biological actions of GSTs are to provide protection against
cellular oxidative stress through neutralization of a wide range of
hydrophobic and electrophilic endogenous compounds or xenobiotics by
catalyzing their conjugation to reduced glutathione. Their biological
functions are extended to include binding protein of steroids
(initially described as ligandin), bilirubin, carcinogens, organic
anions; and peroxidase and [/
5-ketosteroid isomerase
enzymatic activities (6, 8, 9).
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Partial purification of the p28 and p29 and polyclonal
antibody production
Follicles, obtained from cycling cows killed at a local
slaughterhouse, were immediately transported to the laboratory in PBS
(0.6 mM KH2PO4, 4.4 mM
Na2HPO4, 0.15 M NaCl, pH 7.4) on
ice. Granulosa cells were recovered after follicular puncture and
washed in PBS by four centrifugations (500 x g, 10
min, 4 C). Cells were frozen, lyophylized, then resuspended in water
(30 C) and centrifuged (50,000 x g, 1 h, 4 C) to
collect the supernatant soluble proteins. Granulosa cell protein
extracts were precipitated by 25% saturated
(NH4)2SO4 (2 to 4 h at 4 C),
then centrifuged (7,000 x g, 1 h, 4 C). The
supernatant was chromatographed on a phenyl Sepharose CL4B column
(Amersham Pharmacia Biotech, Baie d’Urfé,
Québec, Canada) previously equilibrated with glycine
buffer (20 mM, pH 9) with 25% saturated
(NH4)2SO4. The column was developed
in four steps with glycine buffer of decreasing ionic strength buffer
(25%, 12%, 0% saturated
(NH4)2SO4) and with 50% ethylene
glycol (vol/vol). The four eluted fractions were dialyzed against 5
mM NH4HCO3 pH 8, and then
lyophylized before SDS-PAGE analysis. Each fraction was subjected to
one dimensional SDS-PAGE on a 9–15% gradient slab gel in reducing
buffer conditions as described previously (10). After electrophoresis,
the proteins were either visualized in the gel by Coomassie blue
staining or transferred to a nitrocellulose membrane (11), which was
then stained using red-S-Ponceau. The stained p28 and p29 bands
were cut and used to raise polyclonal antibodies in rabbits
(12).
One dimensional SDS-PAGE and immunoblotting of p28 and p29
Total protein extracts from bovine granulosa cells or corpus
luteum were obtained following homogenization in ice-cold Tris buffer
(60 mM Tris-HCl, 5 mM EDTA, pH 6.8) in the
presence of protease inhibitors (1 mM
phenylmethylsulfonylfluoride (PMSF), 2.5 µg/ml leupeptin, 2.5 µg/ml
aprotinin, 1.2 µg/ml pepstatin-A, 2 mM benzamidine).
Following centrifugation (28,000 x g, 20 min, 4 C),
the supernatant was recovered. Protein concentration was determined
using the Bradford assay (Bio-Rad Laboratories, Inc.,
Hercules, CA) with BSA as standard (Fraction V, Sigma Chemical Co.). Protein extracts (100 µg/well) were incubated
at 100 C for 5 min in reducing conditions then size-fractionated on a
one-dimensional discontinuous SDS-PAGE slab gel (10). After
electrophoresis, the proteins were either visualized after Coomassie
blue staining or transferred overnight at 4 C onto nitrocellulose
membrane (0.45 µm Hybond-C, Amersham) and stained with
red-S-Ponceau. Immunological detection was performed according to
Harlow and Lane (13). Briefly, nonspecific binding sites were blocked
by incubation of the nitrocellulose membrane in a TNT buffer (10
mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween 20)
with 20% normal calf serum (NCS). The membrane was then incubated with
the rabbit polyclonal anti-p28 or anti-p29 antibodies diluted at
1:1,000 in a solution of TNT with 10% NCS. Antigen and first antibody
complexes were revealed following incubation with a second antibody
(goat antirabbit IgG linked to horseradish peroxidase, H & L chains;
human serum adsorbed; Life Technologies, Inc., Burlington,
Ontario, Canada) in TNT buffer with 10% NCS and developed with
4-chloro-1-naphtol (CN) in TN buffer (20 mM Tris-HCl, 150
mM NaCl, pH 7.5).
Immunological screening of the corpus luteum cDNA library
A bovine corpus luteum cDNA expression library was established
to allow the characterization of p28 and p29 since these proteins were
detected in corpus luteum protein extracts. The cDNA library was plated
following commercial protocols (Stratagene, La Jolla,
CA), and the nitrocellulose membranes were then treated with
anti-p28 and anti-p29 as described in immunoblotting section. Positive
clones were purified and screened until all clones were positive.
Positive recombinant phagemid clones were sequenced by the double
stranded dideoxychain termination (14). The consensus nucleotide
sequence of two independent clones (double-stranded sequenced) is
reported. Amino acid sequence was deduced from the nucleotide sequence
and analyzed for sequence homology, hydrophobicity, isoelectric point
and conserved amino acid motifs (15) with programs provided by the
Genetics Computer Group software (version 8, 1994,
Madison, WI).
RNA isolation and Northern analysis of p28 and p29
Northern blot analyses were performed with different bovine
tissues collected at a slaughterhouse as previously described (14). The
RNA molecular weight markers from 9 kb to 0.25 kb (Life Technologies, Inc.) were used to estimate sizes of transcripts.
Total RNA (20 µg) was size-fractionated on agarose gel, transferred
onto a nylon membrane, and hybridized (14) to the full-length 744 bp
(bGSTA1) [32P]-labeled cDNA probe. The membranes were
stripped then rehybridized with the 918 bp (bGSTA2)
[32P]-labeled cDNA probe. The rat elongation factor Tu
cDNA (EFTu; 16) was used as a control gene for RNA expression. The
membranes were exposed for 1 day to photographic film (Biomax-MR,
Eastman Kodak Co., Rochester, NY) at -70 C with an
intensifying screen. The film images were digitized and the intensity
of specific transcripts expressed as ratios of EFTu expression to
account for procedural losses as previously described (14).
Effect of hCG on GSTA1 and GSTA2 expression in bovine ovarian
follicles
Holstein heifers (2 to 3 yr old) were injected on day 7 of the
estrous cycle with 25 mg PGF2 (Lutalyse, Pharmacia & Upjohn, Inc., Kalamazoo, MI) to induce corpus luteum
regression and initiate a follicular phase. Bovine preovulatory
follicles were isolated from eight cows at 0, 6, 12, 18, 20, 22, 24,
and 26 h after the injection of an ovulatory dose of hCG as
previously described (17). Two synchronized heifers received an ovarian
stimulation treatment on day 9 or 10 of the estrous cycle with 320 mg
FSH (Follitropin-V) given in 8 decreasing doses at 12 h interval
over 4 days as described previously (18). They were treated with
PGF2 at the fifth and sixth FSH injection. As for the
previous group of heifers, hCG was injected 36 h following the
first PGF2 treatment. The ovaries were collected at time
0 and 24 h following the hCG treatment. The preovulatory follicles
were dissected from the surrounding ovarian tissue and the follicular
wall recovered and stored individually at -70 C until extracted for
RNA.
Immunohistochemistry
Immunohistochemistry was performed on fixed (PBS buffered 10%
formalin) bovine tissues collected at the slaughterhouse.
Paraffin-embedded tissues were cut at 7 to 8 µm thickness, mounted on
Poly-L-lysine slides, deparaffined through graded xylene
and alcohol series, and rehydrated. Tissues sections were rinsed in TBS
(150 mM NaCl, 0. 1 M Tris, pH 7.5), immersed in
0.1 M glycine in TBS for 10 min, and the antigen epitope
sites were revealed by pressure cooker treatment as previously
described (19). Tissue sections were incubated in TBS with 1% BSA and
1% fat free skim milk to block nonspecific binding sites; then they
were incubated for 16 h at 4 C with the rabbit anti-p28 or
anti-p29 antibody at a dilution of 1:500 in TBS including 1% normal
cow serum. Negative control tissue sections were incubated similarly
with a nonimmune rabbit serum. The first antibody bound to p28 or p29
was revealed with a mouse monoclonal antirabbit IgG conjugated to
alkaline phosphatase (Sigma Chemical Co.) at a dilution of
1:100 for 2 h at room temperature, followed by several washes in
TBS. Visualization was performed using the NBT/BCIP (Roche Molecular Biochemicals, Laval, Québec, Canada)
substrate and photographs taken under bright field illumination.
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). This chromatographic step
represented almost 45% of the total amount of proteins recovered after
phenyl Sepharose chromatography. Following electrophoresis and transfer
onto nitrocellulose, protein bands corresponding to p28 and p29 were
cut and used to raise polyclonal antibodies in rabbits. These
antibodies were shown to bind to proteins of 28 kDa (p28) and 29 kDa
(p29) in supernatant fraction (28,000 x g) of total
protein extracts obtained from bovine granulosa cells and corpus luteum
(Fig. 1B). Attempts to microsequence the NH2-termini of
each protein failed because their N termini were blocked. Therefore,
the characterization of the granulosa cell p28 and p29 was achieved by
screening a 
ZAP bovine corpus luteum cDNA library. The first
immunoscreening of 1.5 x 105 pfu with the anti-p28 or
the anti-p29 antibodies revealed a similar number of positive clones
(0.04%). Two independent clones for p28 and p29 were subsequently
purified to homogeneity, followed by phagemid in vivo
excision and further characterization. Two cDNAs of approximately 850
bp and two cDNAs of approximately 1.0 kb were obtained for p28 and
p29, respectively.
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). The ORF codes for a protein of 222
amino acids of an estimated molecular weight of 25.4 kDa and an
isoelectric point of 8.9. Sequencing the two clones for p29 revealed a
cDNA of 959 bp (Fig. 2B). Analysis of the sequence showed a 5'
untranslated (UTR) region composed of 41 bp, an open reading frame
(ORF) of 666 bp, and a 3'-UTR of 252 bp that included two
polyadenylation signals followed by a polyA+ tail. The ORF
codes for a protein of 222 amino acids of an estimated molecular weight
of 25.6 kDa and an isoelectric point of 7.2. An amino acid homology
search in GenBank for p28 and p29 revealed an overall 82% or 74%
identity to human class glutathione S-transferase (hGSTA; 20),
respectively, and 87% or 78% identity to porcine GSTA (21; Fig. 3). Because these sequences are the first
bovine GSTAs to be characterized, they are named as bGSTA1 and bGSTA2
subunits according to the proposed human glutathione S-transferase
nomenclature (5). Bovine GSTA1 and GSTA2 are 81% identical to each
other and structural differences are mainly located in a 16 amino acid
stretch found between 63M and 81L of bGSTA1
(Fig. 3). Interestingly, bGSTA1 shows the main specific traits of
the class: conserved amino acid required to form a glutathione
binding site (9Y, 15R,45K or
45R, 67Q, 68T, 101D,
131R), amino acids involved in subunit dimerization
(52F, 69R, 73N, 82Y,
89R, 94M, 97E, 98G,
135A, 136F, 139V,
155R), and the motif 68TRAIL72 that
are also present in the human and porcine class GST. However,
bGSTA2 lacks the TRAIL motif that is replaced by a specific 16 amino
acid stretch (64S to 79H) not present in human
and porcine GSTA sequences (Fig. 3). No amino acid match was found for
this specific 16 amino acid GSTA2 peptide when compared by BLAST
analysis in GenBank, underlying the specificity of this new bovine GSTA
isoform subunit. Amino acid consensus motif analysis was performed on
both bGSTAs and showed the presence of three potential serine and
threonine protein kinase C and casein kinase II phosphorylation sites
at position 129TNR, 154SR(K)AD and
202SQR. The potential phosphorylation site
154SR(K)AD is conserved also in human and porcine GSTA.
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).
Analysis of steady-state levels of bGSTA1 and bGSTA2 messenger RNA
(mRNA) showed that some tissues expressed GST at high levels: adult
testis, 7-month old fetal testis, FSH-treated granulosa cells, corpus
luteum, liver, and adrenal gland. Lower levels of bGSTA1 and bGSTA2
expression were observed in lung, thyroid, and kidney, whereas very low
levels were detected in the cotyledon. No expression was found in
epididymis, 3-month old fetal ovary (data not shown), uterus, heart,
spleen, proximal part of duodenum, pyloric region of the stomach, and
the pituitary. A minor mRNA transcript of 3.3 kb for both genes were
observed only in FSH-stimulated granulosa cells, and at a lower level
in the adrenal gland (Figs. 4 and 5B).
Results show that bGSTA1 and bGSTA2 are differentially expressed, and
their level of expression varies tremendously between the tissues
analyzed. The ratio of bGSTA1/bGSTA2 mRNA levels was above 1 in adult
(1.22) and fetal (1.04) testis, granulosa cells (1.2), corpus luteum
(1.15), cotyledon (1.33), adrenal gland (1.31), and kidney (1.28).
However, bGSTA1/bGSTA2 ratios were lower than 1 in the liver (0.74),
the lung (0.55), and the thyroid (0.86), indicating that levels of
expression of the two genes are controlled differently within different
tissues.
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). The expression of bGSTA1 and bGSTA2 in FSH-stimulated
preovulatory follicles was studied in relation to hCG treatment.
Results show a high level of bGSTA1 (1.2 and 3.3 kb) and bGSTA2 (1.4
and 3.3 kb) transcripts in FSH treated follicular wall before hCG (Fig. 5B). Twenty-four hours after the hCG injection, the mRNA levels of
bGSTA1 and bGSTA2 were slightly reduced by 22.2% and 20.2%,
respectively, when compared with follicular wall extract before hCG
injection. The high expression of bGSTA1 and bGSTA2 in FSH stimulated
follicles 24 h following the hCG injection (Fig. 5B) contrasts
with the 77% decrease for bGSTA1 obtained in nonFSH-stimulated
preovulatory follicles (Fig. 5A) 24 h following the hCG injection.
An LH inducible gene, PGHS-2, was used as positive control and showed
expression only at 24 h following the hCG injection (Fig. 5B).
Immunohistochemistry
Immunohistochemical studies were undertaken to identify specific
cell types contributing to GSTA expression. Selection of bovine tissues
analyzed was based on previously observed mRNA expression. In all
bovine tissues analyzed, the use of anti-p28 or anti-p29 gave identical
labeling results. In follicles, GSTA expression was clearly localized
in granulosa and theca cells (Fig. 6, A–H). The signal for GSTA was localized in the cytoplasm of granulosa
cells but not in the nuclei. Granulosa cells of primordial to
preovulatory follicles stained positively. However, heterogeneity in
GSTA staining was observed in granulosa cells of primordial follicles;
some follicles did not stain, whereas others showed a positive signal
(Fig. 6, A and B). In theca cells, a strong GSTA signal was observed in
late preantral, in preovulatory and in late atretic follicles (Fig. 6, C–H). Follicular fluid of large antral follicles showed a positive
reaction. Oocytes from primordial to late antral stages did not express
GSTA. In the corpus luteum, all luteal cells stained positively, but
the intensity of labeling was not uniform. The small luteal cells
showed stronger GSTA signal compared with large luteal cells (Fig. 7, A and B). In the testis, intense
cytoplasmic GSTA staining was associated with Leydig cells. No staining
was observed in cells located within the seminiferous tubules (Fig. 7C). In the kidney, GSTA expression was mainly localized in epithelial
tubular cells with a strong signal in proximal convoluted tubules and
the thick loop of Henle, whereas no staining was observed in the
glomerulus, the distal tubules, and collecting ducts (Fig. 7D). In the
adrenal gland, GSTA was localized in the cortex. Expression of GSTA was
more pronounced in the zona glomerulosa compared with the zona
fasciculata and reticularis (Fig. 7, E and F). The adrenal medulla did
not show expression of GSTA except for groups of chromaffin cells
located near the center of the medulla (Fig. 7, G and H). The
expression of GSTA in the lung was localized in the epithelial cells of
bronchioles with strong staining associated with C-cells cells and
negative staining in the alveoli and stroma (Fig. 7, I and J). In the
liver, the hepatocytes stained uniformly for GSTA, whereas follicular
thyroid cells and cotyledon derived from a 7-month old fetus, showed a
weak GSTA expression (data not shown).
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glutathione
S-transferase subunits (GSTA). We named these subunits bGSTA1 and
bGSTA2, based on the human nomenclature (5). The bGSTA1 and bGSTA2
subunits are 82% and 74% identical to the human GSTA1 subunit (20).
The bGSTA1 subunit shares the 68TRAIL72 amino
acid strech, which is conserved in all known GST class proteins,
and possesses the specific amino acid residues (9Y,
13R, 14G, 20R, 69R,
187R), which are conserved in the , µ, and GSTs.
The GSTA subunit is composed of two domains. Domain 1 spans from the
NH2 terminal to amino acid 86 and from 192 to 222. It
contains the binding site for glutathione, called the G-site, which is
defined by the following conserved amino acids: 9Y,
15R,45K or 45R, 67Q,
68T, 101D, 131R (Fig. 3; 9,
22–26). Domain 2 is less conserved between GSTs and spans from amino
acids 86 to 190. It is involved in the binding of hydrophobic
substrates and is also called the H-site (23, 25). Purification of
GSTAs from bovine adrenal cortex was previously achieved by affinity
chromatography on S-hexylglutathione-Sepharose (S-hex-G-Ag) and their
electrophoretic mobility analyzed by SDS-PAGE (27, 28). However, their
molecular characterization and pattern of expression associated with
cell types in different tissues were not reported. GSTAs were found to
be major cytosolic proteins in the bovine adrenal cortex (28).
Similarly, we have observed a major group of 28 kDa proteins as
demonstrated herein and a previous study (4). In the bovine adrenal
gland study (28), two GSTAs were observed: a major protein band at 25.9
kDa estimated to contribute to 1.3% of the cytosolic protein content,
and a minor protein band at 26.5 kDa. We have shown that bGSTA1
possesses the conserved characteristics of class GST known to allow
interaction with S-hex-G-Ag as reported for other species (22, 29),
which suggest that bGSTA1 may correspond to the 25.9 kDa bovine
GSTA previously reported (28).
The bovine adrenal gland 26.5 kDa GST (28) did not interact to
S-hex-G-Ag but was purified on glutathione-Sepharose (GSH-Ag) affinity
column from material not binding to S-hex-G-Ag matrix. Interestingly,
we have shown that the bGSTA2 subunit presents characteristics not
previously found in class GST: it lacks the TRAIL motif known to be
conserved in GSTA of all species. The TRAIL motif is replaced by a
specific 16 amino acid sequence (64S to 79H)
when compared with bGSTA1, human (20) or porcine class GST (21).
This change in bGSTA2 results in the lost of four recognized amino
acids known to contribute to the G-site and intervene in subunit
dimerization : 67Q and 68T of bGSTA1 contribute
to the G-site by interacting with the 
-glutamyl residues of
glutathione, whereas 69R and 73N are known to
contribute to subunit dimerization. This 16 amino acid peptide
(64S to 79H) is specific to bGSTA2 because no
amino acid match was found when compared with available protein
sequences in GenBank, and thus represents a novel GSTA isoenzyme.
Furthermore, characterization of enzymatic substrate for the two bovine
adrenal GSTs showed a high metabolism of 4-hydroxyalkenal activity when
compared with their human counterpart (28). It was deduced that the
26.5 kDa bovine GST was responsible for the high 4-hydroxyalkenal
activity. The 4-hydroxynonemals, the major endogenously produced form
of 4-hydroxyalkenals, are derived from cellular lipid peroxidation and
are genotoxic (30, 31, 32). Thus, it is reasonable to hypothesize that the
specific amino acid characteristics presented by bGSTA2 could be
responsible for the high 4-hydroxyalkenal processing in the bovine
adrenal gland study (28). Whether bGST2 isoenzyme mediates this
difference remains an interesting and, as yet, open question.
Bovine GSTA1 and GSTA2 mRNA were coexpressed in many bovine tissues as single transcripts of 1.2 kb and 1.4 kb, respectively. Tissues with the highest levels of GSTA mRNA were the ovarian follicle, the corpus luteum, the testis, the liver, and the adrenal gland. Only FSH-treated granulosa cells and the adrenal gland showed the larger 3.3 kb transcript that was observed for both GST isoenzymes. This larger size transcript was found only in tissues with the highest level of GSTA expression. Bovine GSTA1/GSTA2 mRNA ratios varied in different tissues. Bovine GSTA2 mRNA expression was higher than bGSTA1 only in the liver, the lung, and the thyroid. Different steady-state levels of mRNA for bGSTA1 and bGSTA2 indicates tissue-specific and different control of their expression.
The cellular localization of GSTA in different tissues was studied by immunohistochemistry for the first time in the bovine species. In the ovary, GSTA was immunolocalized in the granulosa and theca cells of preantral and antral follicles. Furthermore, GSTA labeling was observed in follicular fluid of large antral follicles, which suggests secretion of GSTA by granulosa cells in the follicular fluid. Heterogeneity in GSTA staining was noticeable in flattened granulosa cells of primordial follicles, which could relate to differences in the physiological stage of follicles (33). GSTA positive follicles could represent the primordial follicles that have entered in the growing pool, whereas GSTA negative primordial follicles could be quiescent follicles. In agreement with our observations, Rahilly et al. (1991) found GSTA expression in human primary follicles, but contrast with the low levels of staining reported for granulosa cells in antral follicles. In the pig (34), no expression of GSTA was observed in granulosa cells contrasting with the bovine and human observations and the recent cloning of two GSTA isoenzymes from an FSH-treated porcine granulosa cell substractive hybridization cDNA library (21). Different levels of GSTA expression reported for granulosa cells may reflect species specific differences, effects of gonadotropins, or differences in the developmental stage at which the follicles were studied. Bovine theca interna cells expressed GSTA. Strong staining was observed concomitant with the beginning of theca interna formation in late preantral follicles up to advanced stages of atresia in large antral follicles. Strong GSTA immunostaining in theca interna cells was also reported in humans (35) and pigs (34). Bovine oocytes at all stages of follicular development showed no expression of GSTA.
A significant reduction (77%) in the expression of GSTA mRNA was observed in preovulatory follicles isolated 12 h post-hCG injection, which agrees with the observed decrease synthesis of the 28–30 kDa protein group in granulosa cells after the LH surge (4). Using the same animal model, it was previously shown that estradiol concentrations were reduced in follicular fluid following hCG injection (17, 18). Therefore, reduction in GSTA mRNA expression was concomitant with the reduction of estradiol concentrations. However, when we pretreated follicles with FSH, they responded differently to hCG with only a slight decrease (21%) of expression for GSTAs mRNA at 24 h post-hCG injection. It is well known that ovarian stimulation with FSH in cattle increases steroid synthesis as found in follicular fluid (36). Corroborating our observations, gonadotropin treatment of luteinizing porcine granulosa cells (34) or rat ovary (37) was shown to increased GSTA expression. In the bovine model, prostaglandin endoperoxidase synthetase-2 (PGHS-2) mRNA was induced between 18 to 26 h post-hCG injection (17, 18). PGHS-2 is the first rate limiting enzyme in the prostaglandin biosynthesis pathway, and prostaglandins are required for the ovulatory process. Collectively, these results suggest that GSTA mRNA expression is not temporally related to PGHS-2 expression but is modulated by gonadotropins and intimately linked to estradiol synthesis.
The heterogeneity of immunohistochemical localization in bovine corpora lutea was related to a stronger expression of GSTA in small compared with large luteal cells, which confirms observations in human and porcine corpus luteum (34, 35). In cattle, granulosa cells differentiate into large luteal cells, whereas theca interna cells differentiate into small luteal cells (38). The large luteal cells produce high basal concentrations of progesterone but are not responsive to LH, whereas small luteal cells produce low basal levels of progesterone but are stimulated by LH. The significance of high GSTA expression in small luteal cells is unknown but suggests a potential association with steroidogenesis and LH. In the bovine testis, strong staining for GSTA was found in the testosterone-producing Leydig cells, and no expression was detected in all cells of the seminiferous tubule. These observations agree with those in humans (39). We did not observe GSTA1 nor GSTA2 mRNA expression in the bovine gastrointestinal tract (stomach and intestine) except for expression in the liver, which contradicts observations in human gastrointestinal tissues (39). Bovine adrenal cortex showed strong expression of GSTA in the zona glomerulosa compared with the zona fasciculata and reticularis, in contrast to very low levels observed in human zona glomerulosa and high levels in the zona reticularis (39). Staining for GSTA was observed in the zona glomerulosa and fasciculata of the rat adrenal gland (40). Negative staining was observed in most of the bovine adrenal medulla except for islands of chromaffin cells, which showed a strong signal, confirming observations in rat adrenal medulla (40). In the bovine kidney, GSTA labeling was associated only with epithelial cells in proximal convoluted tubules and the thick descending loop of Henle, agreeing with observations reported in human (39). In agreement with data in cattle (41) and humans (42), we found GSTA mRNA expression in bovine lungs. These observations contradict previous reports that found no GSTA subunit expression in bovine lung (28). Moreover, we found strong GSTA immunostaining in bovine bronchioles that was associated exclusively with Clara cells. Our observations in the bovine species differ from the immunostaining for GSTA in humans where intensity of staining was low but widely distributed, extending from the epithelium of the bronchi to the type 1 and type 2 alveolar epithelium, and to vascular smooth muscle (42). The differences observed in the expression of GSTA in the bovine and human species may be associated with cross-reaction of antibodies used in the human study or to species specific difference in GSTA expression.
The common biochemical link shared by the different cell types that supports the expression of GTSAs remains speculative. The gonadal, adrenal, and liver cells contain mitochondrial and microsomal associated cytochrome P450 enzymes involved in steroidogenesis (43). Renal epithelial tubular cells are known to be involved in the synthesis of vitamin D (43). The human or rat lung were shown to have the capacity to metabolize steroid hormones (44, 45), and Clara cells express specific cytochrome P450 involved in the metabolism of xenobiotics (46). Steroid hormones synthesis and metabolism was shown to release free radicals (47). These free radicals may thereby stimulate GSTA expression (31, 32). Expression of GSTA isoenzymes in bovine species may thus be linked to cell types involved in steroid synthesis or metabolism, although validation of this hypothesis will require future work. In conclusion, this report is the first to demonstrate in the bovine species that expression of GSTA isoenzymes is tissue and cell specific and is modulated by gonadotropins in the developing bovine ovarian follicle. We have also shown that the highest level of GSTA expression was localized to steroidogenically active cells. The specific biological role of GSTA may be linked to cellular oxidative stress induced by reactive oxygen species generated during steroidogenesis.
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Acknowledgments |
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Received November 19, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1–1 GST.
J Biol Chem 268:19188–19191-class
glutathione transferase A1–1 in the apo-form and in complexes with
ethacrynic acid and its glutathione conjugate. Structure 3:717–727[Medline]
glutathione
transferase A1–1. J Mol Biol 247:765–773[CrossRef][Medline]
-class glutathione S-transferase in bovine and human
adrenal cortex tissues. J Endocrinol 132:83–92 and p class glutathione
transferases in normal human tissues. Pharmacol Toxicol 72:321–331[Medline]
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