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Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Lisa M. Olson, Ph.D., Department of Obstetrics and Gynecology, Box 8064, Washington University School of Medicine, 4911 Barnes Hospital Plaza, St. Louis, Missouri 63110. E-mail: olsonl{at}kids.wustl.edu
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NO is synthesized by rat and human ovarian cells and has been shown to influence steroidogenesis, ovulation rate, and ovarian apoptotic cell death (7, 8, 9, 10, 11, 12). We have identified endothelial NOS (eNOS) and iNOS in rat luteinized ovaries and have shown that NO negatively regulates luteal estradiol synthesis in vitro (13). NO also has been suggested to play a role in ovulation. Rodents treated with pharmacological inhibitors of NOS show a reduced ovulation rate relative to controls, and NO has been proposed to function in ovulatory inflammatory events (7, 9, 10, 12, 14). However, NO also has been shown to lower follicular apoptotic cell death, which may independently influence ovulation rate (11). Recently, the expression of the messenger RNAs (mRNAs) for eNOS and iNOS in the rat ovary have been shown to be regulated during follicular development (15).
In the present study, we examined the expression of NOS proteins in the rat ovary during follicular development, ovulation, and pseudopregnancy. Using isoform-specific antisera, we localized eNOS and iNOS in the rat ovary. We found that both the constitutive endothelial and inducible isoforms of NOS show distinct cell-specific expression patterns and exhibit regulation by the hormonal signals that direct ovarian development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and tissue preparation
Immature female Sprague-Dawley rats (24 days old) were obtained
from SASCO (Omaha, NE). Animals were housed in a 25 C room with a 14-h
light, 10-h dark cycle. The rats received rat chow and tap water
ad libitum. Immature rats (25 days old) were given either an
sc injection of saline (control) or 10 IU of PMSG. Forty eight hours
later, the PMSG-treated rats were injected sc with 10 IU of hCG (27
days old). These treatments cause a large cohort of follicles to
undergo sequentially follicular development, ovulation, luteinization,
pseudopregnancy, and luteolysis (17, 18). Pseudopregnancy lasts
approximately 10 days, and physiologically mimics the first half of a
rat gestation (17, 18). The control group of rats were killed on days
25, 26, and 27 of age. Two treatment groups were killed 24 h and
48 h after PMSG treatment (on days 26 and 27 of age; follicular
development). The remaining rats were killed 2 h, 8 h,
12 h, 20 h or 6 days and 10 days after hCG injection
(ovulation followed by luteinization). Ovaries were removed and either
immediately snap-frozen in liquid nitrogen or placed in Tissue-Tek OCT
embedding compound and frozen in liquid nitrogen. Frozen specimens were
stored at -80 C until sectioned for immunohistochemistry or used for
Western blot analysis. Each group of rats consisted of 3–5 animals,
and both ovaries from each rat were examined. All animals were killed
with 100% CO2 followed by cervical dislocation.
The animal protocol used was approved by the Washington University Medical School institutional committee on laboratory animal care and was conducted in accordance with the NIH guide for the care and use of laboratory animals.
Immunofluorescent staining
The immunostaining procedure was performed by a modified
immunofluorescence technique (13, 19). Cryosections (6 µm in
thickness) were cut at -17 C on a Reichert-Jung cryostat (Leica,
Heilderberg, Germany) and mounted on microscopic slides coated with
poly-L-lysine (Sigma Chemical Co.). After 30 min at 25 C,
slides were fixed in Carnoy’s solution (ethanol-chloroform-glacial
acetic acid, 6:3:1, for bNOS and eNOS stainings) or in freshly prepared
1% paraformaldehyde solution (for iNOS staining) in PBS (0.01
M phosphate, 0.14 M NaCL; pH 7.4). After
washing twice with distilled water for 5 min each time, slides were
treated with 0.3% hydrogen peroxide in absolute methanol for 30 min to
quench endogenous peroxidase activity. Then the slides were washed
twice in distilled water and twice in PBS containing 0.3% Triton
X-100. All subsequent incubations with immunochemicals were performed
in a humidified chamber.
To reduce nonspecific binding, blocking buffer (PBS containing 5% normal horse serum for monoclonal antibody or PBS with 5% normal goat serum for polyclonal antisera) with avidin solution (blocking kit; 4 drops/ml of buffer) was applied to slides for 60 min at 25 C. Sections were incubated either with a bNOS monoclonal mouse antibody, eNOS, or iNOS polyclonal rabbit antisera, each diluted 1:100 in blocking buffer with biotin solution (blocking kit; 4 drops/ml of buffer) overnight at 4 C. After four washes with PBS, slides were incubated with biotinylated horse antimouse IgG (1:100 dilution in blocking buffer) or biotinylated goat antirabbit IgG (1:200 dilution in blocking buffer) for 90 min at 25 C. After washes in PBS, the final detection step was performed for 1 h at 25 C in the dark with fluorescein-isothiocyanate-avidin (1:20 dilution in PBS). After this incubation, the slides were washed four times in PBS and subsequently counterstained with PI (1:500 dilution in PBS) to visualize all cellular nuclei. After three washes in PBS, slides were mounted in Vectashield mounting medium.
For each primary antibody (bNOS, eNOS, and iNOS) the immunostaining procedure was conducted on several sections from each ovary. Several negative control sections per ovary were incubated with control ascites fluid in place of the monoclonal antibody, or normal rabbit serum or rabbit IgG in place of polyclonal antisera. An additional negative control was conducted for the eNOS polyclonal rabbit antiserum obtained from Monsanto/G.D. Searle (16). To confirm that eNOS immunofluorescent localization was specific, a blocking assay was conducted in which the primary antiserum was preincubated with an excess of the antigen peptide (50 µg/ml) for 4 h at 25 C. This preincubated antiserum solution was used for immunostaining as described above.
Incident light fluorescence was monitored with a Nikon light microscope equipped with a mercury lamp and a dual wavelength filter (Boyce Scientific, St. Louis, MO) to permit the simultaneous detection of green and red fluorescence. Ektachrome 400 film (Eastman Kodak, Rochester, NY) was used for photomicroscopy.
Western blot analysis for eNOS and iNOS
To quantitate changes in protein levels, Western blot analysis
of ovarian homogenates was performed according to the procedure
described by Olson et al. (13, 20). Because rat ovaries did
not show any positive immunostaining for bNOS at any of the time points
examined (data not presented), Western blot analysis was performed only
for eNOS and iNOS.
Briefly, frozen rat ovaries from control rats (no PMSG; 27 days old), from rats 48 h after PMSG treatment (27 days old), and 12 h, 20 h, and 10 days after hCG treatment, were placed in 300 µl of ice-cold homogenization buffer (50 mM Tris-base, pH 7.4; 10 mM EDTA; 150 mM NaCl; and 0.1% Tween-20 with the addition of 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1% ß-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml soybean trypsin inhibitor), homogenized on ice with a Micro Tissue Grinder (Kontes, Vineland, NJ) and centrifuged at 25,000 x g for 30 min at 4 C. The protein content of the resulting supernatant was determined using BSA as the standard (21). Equal amounts of protein (65 µg) from homogenized ovaries were diluted in 6x SDS sample buffer (0.125 M Tris-Cl, pH 6.8; 10% SDS; 30% glycerol; 9.3% dithithreitol; and 0.012% bromophenol blue dye) and heated at 95 C for 4 min. Protein fractions were loaded onto 4–20% gradient SDS-PAGE gels and electrophoretically separated for 2 h at constant current (30 mA) (22). Rainbow-colored protein molecular mass markers (14.3–200 kDa) were always run on each gel. Separated proteins were electrotransferred onto Immobilon-P membranes using a Mini Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) at 4 C overnight at constant voltage (30 V) in a buffer containing 20% methanol, 25 mM Tris, 192 mM glycine, and 0.02% SDS. Then the membranes were washed in 0.05% Tween-20 in a solution of 137 mM NaCl and 20 mM Tris at pH 7.5 (T-TBS) and incubated in blocking buffer (5% milk powder in T-TBS) for 1 h at 25 C. The presence of eNOS or iNOS was confirmed by incubating the membranes in blocking buffer containing either eNOS or iNOS monoclonal antibody at a dilution of 1:2,500 and 1:1,000, respectively. After washing in T-TBS, immunoblots were incubated with a HRP-conjugated antimouse IgG (1:5,000 dilution for eNOS or 1:3,500 dilution for iNOS in blocking buffer) for 1 h at 25 C. Finally, the NOS isoforms were detected using the ECL detection kit. The relative levels of eNOS and iNOS in rat ovaries were quantified by densitometric scanning of the exposed films from Western blots (n = 5 rats for eNOS and n = 3 rats for iNOS in each treatment group) using Molecular Dynamics Image Quant Software on a Personal Densitometer (Molecular Dynamics, Sunnyville, CA).
Several cell lines and rat tissues were used as positive and negative control samples on eNOS and iNOS Western blots. Positive controls for eNOS included homogenates of bovine endothelial cells (23) and rat lung and uterus (collected 20 h after hCG injection) (24). Samples that were expected to be negative for eNOS were homogenates of LPS-activated (10 µg/ml for 10 h) RAW 264.7 macrophages and rat cerebellum (obtained 20 h after hCG injection) (23, 25). Positive controls for iNOS included homogenates of LPS-activated RAW 264.7 macrophages (26), and liver and spleen homogenates from rats injected with LPS (2 mg/kg BW, ip for 6 h) (27, 28). The homogenate from bovine endothelial cells was included as a negative control for iNOS (25).
To verify equal protein loading, Immobilon-P membranes (before immunodetection of eNOS and iNOS) always were stained with Ponceau S and washed with distilled water. In addition, to determine the specificity of changes in eNOS and iNOS expression, Western blots for GAPDH were conducted according to the method described above. We expected the level of GAPDH, often considered a housekeeping protein, to remain constant during follicular and luteal development. Immobilon-P membranes that had been used for eNOS and iNOS immunodetections were stripped of bound antisera (n = 2) and reprobed with a GAPDH monoclonal antibody at a dilution of 1:300. After washing in T-TBS, immunoblots were incubated with a HRP-conjugated antimouse IgG at the dilution of 1:2,500 and detected using the ECL detection kit.
Statistical analysis
One-way ANOVA was used to analyze the changes in eNOS and iNOS
protein levels during follicular development, ovulation, and
pseudopregnancy, followed by linear contrasts to determine significance
between means (16). The relative expressions of eNOS and iNOS were
calculated by dividing the protein levels in each treatment group by
the protein level in the control group (no PMSG; 27-day-old rats).
P < 0.05 was considered to be significant. Data were
analyzed using the Number Cruncher Statistical System Software version
5.03 (NCSS, Oakland, CA) (29).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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for an ovary from a
27-day-old rat). PI counterstaining identified all cell nuclei, as
indicated by red fluorescence. No specific staining was
found within the granulosa cell layer (Fig. 1A). All three control
groups showed the same pattern of ovarian eNOS staining. After
stimulation of follicular development with PMSG for 24 h or
48 h (26- and 27-day-old rats, respectively), many large antral
follicles with well-delineated, multiple granulosa and theca cell
layers were present (Fig. 1B for 48 h; data not presented for
24 h). The cytoplasm of cells in the theca layer and scattered
granulosa cells showed strong positive green
immunofluorescent staining, suggesting that PMSG induced eNOS
expression in these cells (Fig. 1B). After injection of hCG (2 h,
8 h, and 12 h), follicles containing strong green
immunofluorescence in both theca and granulosa cell layers remained,
indicating the continued presence of eNOS protein (Fig. 1C for 12
h; data not presented for 2 h and 8 h). Within granulosa
cells, a heterogenous pattern of staining was observed. Mural granulosa
cells (located near the basal lamina) showed strong positive staining
for eNOS, whereas antral granulosa cells (located near the follicle
antrum) were negative (Fig. 1C). This staining pattern shows that hCG
administration resulted in expression of eNOS within specific granulosa
cell subtypes. After ovulation and luteinization, 20 h post-hCG
injection, immunoreactivity for eNOS was seen as punctate cytoplasmic
staining in luteal parenchymal cells of the developing corpus luteum CL
(Fig. 1D). In well-established CL (6 days after hCG injection; data not
presented) and in degenerating CL (10 days after hCG injection),
intensely stained luteal parenchymal and nonparenchymal cells
(e.g. endothelial, fibroblast) were observed, and the
distribution of staining seemed uniform throughout the entire CL (Fig. 1E). Control slides for each ovary were stained with normal rabbit
serum or rabbit IgG instead of primary antiserum. An example of a
control slide from an ovary obtained 48 h after PMSG injection is
shown in Fig. 1F. No immunofluorescent staining is observed. All other
negative control slides at each stage of ovarian development also were
negative (data not presented). In addition, no apparent staining was
observed when the primary eNOS antiserum was preincubated with an
excess of the antigen peptide (data not presented).
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Western blot analysis of eNOS in rat ovaries
To quantitate the changes in eNOS expression during follicular
development, ovulation, and pseudopregnancy, relative eNOS protein
levels were determined by Western blot analysis (Fig. 2). We observed a protein of approximately 135 kDa [the
molecular mass of eNOS (23, 30, 31, 32, 33)] in bovine endothelial cells (Fig. 2A, lane 1), ovaries obtained from control rats (no PMSG; 27 days old)
or 48 h after PMSG injection (27 days old) and 12 h, 20
h, and 10 days after hCG injection (Fig. 2A, lanes 2–6), and in rat
uterus and lung (Fig. 2A, lanes 11–12). We did not detect eNOS in
homogenates of LPS-activated RAW 264.7 macrophages (Fig. 2A, lane 10)
or rat cerebellum (Fig. 2A, lane 13). In addition, we failed to detect
eNOS in ovarian homogenates when eNOS antibody was omitted (Fig. 2A, lanes 7–9 are representative controls for lanes 4–6,
respectively).
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. During PMSG-induced follicular growth, eNOS
levels increased 2.5-fold relative to control ovaries
(P < 0.05). eNOS levels were further increased 12
h and 20 h after hCG injection, compared with control ovaries
(5-fold and 7-fold, respectively; P < 0.05). However,
the greatest amount of eNOS was observed in ovaries 10 days after hCG
injection, which contained 15-fold greater eNOS levels than control
ovaries (P < 0.05). There were no changes in
expression of GAPDH during hormone-directed ovarian differentiation
(Fig. 5). These data indicate that equal amounts of protein were loaded
and support the specificity of the eNOS response to gonadotropin
treatment. These quantitative data are consistent with the changes in
eNOS expression observed immunohistochemically and demonstrate that in
the ovary, eNOS is not a constitutively expressed enzyme. Indeed,
ovarian eNOS exhibits a distinct, hormone-regulated expression pattern
during follicular development, ovulation, and pseudopregnancy in this
rat model.
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). In ovaries
obtained from control rats (no PMSG; 25, 26, or 27 days old), we found
iNOS expressed within the theca cell layer and stroma surrounding
immature follicles (Fig. 3A; yellow-green fluorescence for
an ovary from a 27-day-old rat). All three control groups showed the
same pattern of ovarian iNOS staining. When follicular development was
stimulated with PMSG for 24 h (data not presented) or 48 h
(27-day-old rats), we saw a slight increase in iNOS within the theca
cell layer and stroma (Fig. 3B). In contrast to eNOS expression, we did
not observe any staining within granulosa cells or on the surface of
oocytes. Thus, the cellular expression pattern of iNOS and eNOS are
distinct. Furthermore, unlike the eNOS isoform, iNOS is not induced
during follicular development. After hCG injection (2 h, 8 h, and
12 h), strong positive immunofluorescent staining was observed
within the theca cell layer of preovulatory and ovulatory follicles and
the interstitial cells surrounding them (Fig. 3C for 12 h; data
not presented for 2 h and 8 h). At 20 h after hCG
injection (about 8 h after ovulation), iNOS immunoreactivity had
decreased and was observed only in cells of external layers of the
developing CL (Fig. 3D). In degenerating CL (10 days after hCG
injection; Fig. 3E), iNOS positive staining was expressed in
nonparenchymal cells within the CL. An example of a negative control
section (ovary obtained 10 days post-hCG) incubated with normal rabbit
serum, instead of iNOS antiserum, shows no immunofluorescent staining
(Fig. 3F). All other control slides at each stage of ovarian
development also were negative (data not presented).
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Western blot analysis for iNOS in rat ovaries
On the basis of immunostaining, levels of iNOS seemed to be
hormonally regulated. We confirmed the presence of iNOS in the rat
ovary using Western blot analysis with an iNOS-specific monoclonal
antibody (Fig. 4). We detected a protein migrating at
approximately 130 kDa [the molecular mass of iNOS has been reported to
vary between 130–150 kDa (4, 25, 32, 33, 34)] in homogenates of
LPS-activated RAW 264.7 macrophages (Fig. 4A, lane 1), ovarian
homogenates from control rats (no PMSG; 27 days old) or 48 h after
PMSG injection (27-day-old rats) and 12 h, 20 h, and 10 days
after hCG injection (Fig. 4A, lanes 2–6), and homogenates of liver and
spleen from a LPS-injected rats (Fig. 4A, lanes 10–11). We did not
detect iNOS protein in bovine endothelial cells (Fig. 4A, lane 9).
Ovarian homogenates also were negative when iNOS antibody was omitted
(Fig. 4A, lanes 7 and 8 are representative controls for lanes 5 and 6,
respectively). For ovaries from rats 12 h after hCG injection, a
second band of greater molecular mass also was detected. This protein
of approximately 150 kDa also has been identified in activated
macrophages (25, 26, 32, 35), in kidney from LPS-injected rats (27), in
human ovarian tumors (36), and in COS-7 cells transfected with eNOS
complementary DNA, where it was thought to represent a
posttranscriptionally modified form of eNOS (30).
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). Expression of iNOS was low in
ovaries from control rats and was not significantly increased during
PMSG-induced follicular development. In contrast, hCG injection
increased the level of ovarian iNOS by 6-fold relative to control rats
(P < 0.05). As luteinization progressed (20 h after
hCG injection) the iNOS band intensity was 7-fold higher than control
ovaries (P < 0.05) but was not significantly higher
than the previous treatment group. However, iNOS levels increased
10-fold above control by 10 days after hCG injection (P
< 0.05). The lack of regulation of GAPDH levels during these ovarian
stages supports the specificity of the iNOS response (Fig. 5). These quantitative data strengthen our immunostaining results and demonstrate that ovarian iNOS is significantly increased during hCG-induced ovulation in rats.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The various isoforms of NOS often are named after the cell in which the enzyme was first described (e.g. eNOS was first discovered in the endothelium); however, NOS isoforms have been described in a variety of cell types and in several species (2, 23, 37). eNOS is a membrane-bound protein of 135 kDa that is synthesized by endothelial cells and other cell types in several bovine, human, porcine, and rodent tissues (adrenal gland, kidney, lung, ovary, pancreatic islets, placenta, uterus) (8, 12, 15, 16, 23, 24, 38, 39, 40). The immunohistochemical localization of eNOS in the present study indicates that this enzyme also is present in mural granulosa cells, theca cell layer and ovarian stroma of antral follicles, and luteal parenchymal and nonparenchymal cells of the CL in the rat ovary. The present results extend our previous studies that demonstrated eNOS expression by endothelial and steroidogenic cells of luteinized ovaries (13). Our findings also are in agreement with other studies that have localized eNOS within the endothelium of the ovarian vasculature (12, 15), theca interna of preovulatory follicles in rodents (12, 40), and in human granulosa-luteal cells (8).
In the present study, we have documented hormonal regulation of ovarian
eNOS. The expression of eNOS increased after PMSG-induced follicular
development and continued to rise after an ovulatory dose of hCG to
reach its maximum expression in the CL. Our observations concerning
hormonal regulation of eNOS protein support those of Van Voorhis
et al. (15), who observed an increase in eNOS mRNA after
gonadotropin stimulation of immature rats. eNOS is commonly described
as a constitutively expressed protein, but its levels can be altered by
various stimuli, including shear-stress, tumor necrosis factor-
, and
estrogen (35, 38, 41). Our results demonstrate that, in the ovary, eNOS
protein levels are regulated by hormones during ovarian follicular
development and luteinization.
iNOS (130–135 kDa protein) was first isolated and characterized from activated murine macrophages (4, 42). Increased expression of iNOS in other cell types has been correlated with a number of pathological situations such as septic shock, stroke, and diabetes (1, 37, 43, 44). Studies of iNOS expression during normal physiology have been less frequent; however, exciting new evidence points to expression of iNOS in response to signals that are noninflammatory or nonimmunologic (45, 46, 47). Reported examples of tissues normally expressing iNOS include the fallopian tubes, kidney, thymus, and uterus in the rat, mouse uterus, pregnant rabbit uterus, and large airways in humans (35, 44, 45, 46, 47, 48). In the present investigation, we have demonstrated that the rat ovary also expresses iNOS protein, which responds to gonadotropin stimulation of normal follicular development. Using immunocytochemistry, we localized iNOS in the theca cell layer and ovarian stroma of antral follicles and nonparenchymal cells of the CL. In contrast to results with eNOS, granulosa cells were devoid of staining. These results extend our previous studies that showed that iNOS is expressed in luteal cells located around the blood vessel network within the CL (13). Our data concerning the location of iNOS protein differs from a previous report demonstrating expression of iNOS mRNA in granulosa cells of secondary preantral follicles (15).
Our immunostaining results were confirmed by Western blot analysis, which indicated the presence of iNOS protein in the rat ovary. The levels of iNOS were regulated by gonadotropins, albeit in a different manner from that observed with eNOS. During PMSG-induced follicular development, iNOS protein levels remained relatively constant. An ovulatory dose of hCG resulted in an increase in iNOS protein that reached its maximum level in the late CL. Others have reported maximal expression of iNOS mRNA levels in unstimulated ovaries that were reduced after hCG injection (15). Thus, hCG administration results in decreased iNOS mRNA, whereas iNOS protein levels continue to rise. These data suggest that hCG may facilitate the translation of iNOS mRNA into protein. A similar pattern of regulation of eNOS mRNA and protein by hypoxia has been reported in endothelial cells (41).
Several lines of evidence support multiple roles for NO within the ovary. NO has been shown to negatively influence estradiol synthesis in both rat and human luteal cells in vitro (8, 13). NO has been implicated also in the gonadotropin regulation of an ovarian blood-follicle barrier, which has been shown to exist at the level of the ovarian microvasculature (12). NO has been suggested to act as a potent and fast-acting signal that regulates the barrier’s ability to block the entrance of certain blood components into the follicular fluid (12). Our observations that eNOS and iNOS are expressed in theca cell layer and stroma cells of preovulatory follicles support this possible role for ovarian NO.
The ovarian NO/NOS system also has been suggested to function in ovulation and follicle rupture through its known effects on vascular dilation and ovulatory leukocyte distribution (10, 14, 49, 50). It also has been demonstrated that pharmacological inhibitors of NOS suppress hCG-induced ovulation in rodents (10, 12). The specificity of this effect was confirmed by the ability of a NO generator to reverse the inhibitory action (10). We observe an induction of both eNOS and iNOS with an ovulatory dose of hCG, and thus our data certainly support a role for increased NO production during ovulation. NO also has been shown to reduce follicular apoptosis in ovaries (11), which could explain a reduction in ovulation rate with NOS inhibitors (7, 9, 10, 12, 14).
In summary, we have demonstrated that eNOS and iNOS are present in the rat ovary. During ovarian follicular development, they respond to gonadotropin stimulation with distinct cell-specific patterns of expression. The goal of future studies is to understand the unique function of each NOS isoform in ovarian physiology.
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Acknowledgments |
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Footnotes |
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Received June 17, 1996.
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K. Mitsube, U. Zackrisson, and M. Brannstrom< |
