2. charge-selective blood follicle barrier (BFB), which is lo-
cated within the endothelium of the vascularized theca (8).
With an ovulatory surge, LH binds to LH receptors in this
endothelium and increases vascular permeability of the BFB
through NO (9). The BFB opens to allow the entry of serum
glycoproteins belonging to the inter-␣-inhibitor (I␣I) family
that bind to hyaluronic acid, stabilizing the extracellular
matrix between cumulus cells and the oocyte to facilitate
ovulation (10).
Studies in humans have also provided evidence for the
involvement of NO in PCOS. Nitric oxide levels have been
positively correlated with follicular size suggesting that NO,
in part, may regulate whether follicles become cystic or
remain normal (11). In addition, blood flow studies have
demonstrated that ovarian stromal blood flow is higher in
PCOS patients (5, 12). Therefore, follicular cysts and high
blood flow may be attributed to elevated NO in COD/PCOS
patients. Furthermore, during ovulation induction protocols,
supplementation of L-arginine, the amino acid from which
NO is synthesized, improves follicular blood flow and ovar-
ian responses (13). Thus, these studies have begun to eluci-
date the role of NO in follicular cyst formation and treat-
ment.
These data correlating NO in PCOS patients combined
with what is known about ovarian BFB function led us to
hypothesize that increased concentrations of circulating LH
causes elevated NO, initiating a defect in the BFB leading to
follicular cysts. To test this hypothesis, we induced cystic
development in immature mice by implanting miniosmotic
pumps that deliver and maintain constant levels of hCG.
Within 7 days, these mice displayed cystic follicles with
associated complications such as increased T levels, altered
ovulation patterns, and impaired BFB response. In these
mice, chronic infusion of the nitric oxide synthase (NOS)
inhibitor NG
-nitro-L-arginine methyl ester (L-NAME) during
cystic formation resulted in a reduction of cyst size, main-
tenance of normal T levels, and intact BFB. Our results also
demonstrate that hCG-induced follicular cysts and their as-
sociated complications can be ameliorated by the NOS in-
hibitor L-NAME, providing support for the involvement of
NO in follicular cyst pathogenesis.
MATERIALS AND METHODS
Animals
Immature female B6D2F1 mice (23 Ϯ 2 days old) were
purchased from Taconic Farms (Germantown, NY). Animals
were housed in static microisolator cages, fed commercially
available rodent chow (Harlan Teklad LM 485 Mouse/Rat
Sterilizable Diet, Indianapolis, IN), and provided sterilized
tap water ad libitum during a 12/12-hour light/dark cycle
with no twilight. All experimental procedures were per-
formed at age 25 Ϯ 2 days with the approval of the Univer-
sity of Cincinnati Institutional Animal Care and Use Com-
mittee.
Animal Experiments
Before surgery Alzet osmotic minipumps (models 1003D
and 1007D; Alza Corp., Palo Alto, CA) were filled with
hCG, L-NAME, or NG
-nitro-D-arginine methyl ester (D-
NAME) (Sigma Chemical Co., St. Louis, MO) dissolved in
sterile filtered deionized water and primed according to
manufacturer’s instructions. Mice were anesthetized with
metofane (methoxyflurane; Schering-Plough Animal Health
Corp, Union, NJ) inhalant. An intraperitoneal (IP) injection
of 5 IU pregnant mare serum gonadotropin (PMSG; Profes-
sional Compounding Centers of America, Houston, TX) was
administered followed by insertion of the hCG containing
osmotic minipump into the subcutaneous space between the
scapulae. Minipumps allowed continuous infusion of 250
mIU/h hCG. This dose was based on the previously de-
scribed transgenic mice by Risma et al. (14) in which LH is
overexpressed and mice display infertility and polycystic
ovaries. For double pump experiments, two pumps were
inserted into the subcutaneous space between the scapulae in
either a combination of hCG and L-NAME (hCG ϩ L-
NAME) or hCG and D-NAME (hCG ϩ D-NAME) in
PMSG-primed mice. Minipumps allowed continuous simul-
taneous infusion of 250 mIU/h hCG and 3 mg/kg/h L-
NAME or D-NAME. The dose of L-NAME was determined
from a dosage study comparing 3, 6, and 9-mg/kg/h using
gross and histological ovarian morphology as a marker.
The hCG-treated mice were euthanized every day during
a period of 7 days for tissue collection. On the basis of
data from hCG-only treated mice indicating that the most
significant changes occurred on days 2, 3, and 6, double
pump-treated mice were euthanized on those days. Sham-
operated animals were treated identically with the excep-
tion of pump insertion; initially, pilot studies using pumps
filled with phosphate-buffered saline (PBS) were tested.
Blood (approximately 0.5 mL) was collected into ethyl-
enediaminetetraacetic acid (EDTA)-treated tubes by car-
diac puncture resulting in plasma. Ovaries and cumulus
oocyte complexes (COCs) were collected by dissection.
Hormone Analysis
Plasma was collected from treated and sham-operated
mice on indicated days after implantation surgery. Hormone
levels (intact hCG, hFSH, and free T) were determined by
using the appropriate ELISA or enzyme immunoassay assay
kits according to manufacturer’s instructions (Diagnostic
Systems Laboratories, Houston, TX).
Histology and Immunohistochemistry
Frozen sections (8 m) of 4% paraformaldehyde-fixed
and 30% sucrose-infused ovaries were prepared on a cryostat
(Leitz, Wetlar, Germany). Sections were treated with either
hematoxylin and eosin (H & E), or immunohistochemical
protocols. For immunohistochemistry, sections were blocked
in immunohistochemistry buffer (5% goat serum, 5% bovine
serum albumin/PBS), incubated with primary antibody, rab-
1302 Nemade et al. NO in murine ovarian follicular cysts Vol. 78, No. 6, December 2002
3. bit antihuman (I␣I) (1:500; Dako Corp., Carpinteria, CA)
and secondary antibody, Alexa 488-conjugated goat antirab-
bit IgG (1:1,000; Molecular Probes, Eugene, OR). Speci-
mens were examined using a Nikon fluorescent microscope
(Microphot-FX, Nikon, Japan) and digitally photographed
using a Spot2 camera (Diagnostic Instruments, Sterling
Heights, MI).
Follicular Measurements
The Metamorph Imaging System (Universal Imaging
Corp, West Chester, PA) was used to measure the perimeter
of randomly selected ovarian follicles using the basement
membranes as a guide.
Ovulation Assays
Both sham-operated and treated mice were superovulated
on indicated days with 5 IU hCG IP injections. After 16
hours, mice were euthanized and the number of COCs ovu-
lated into the ampoule were counted.
Blood Follicle Barrier Experiments
Negative control animals were treated with an injection of
5 IU IP PMSG and euthanized 48 hours later for ovary
collection. Positive control animals were primed with PMSG
as described and followed by 5 IU IP of hCG. Treated mice
were given a single injection of 5 IU IP of hCG (experimen-
tal ovulatory surge) or an IP injection of 2.5 mg/kg of
sodium nitroprusside (Sigma Chemical) on indicated days.
Mice that received either hCG or sodium nitroprusside were
euthanized after 6 hours and ovaries collected for immuno-
histochemical analysis of the blood follicle barrier as previ-
ously described (9).
Statistical Analysis
Data reported are expressed as the mean Ϯ SEM or mean
Ϯ confidence interval. For hormone analysis, statistical sig-
nificance was determined by using Student’s t-test in Mi-
crosoft Excel Data Analysis Software (Microsoft Corp., Se-
attle, WA). For complicated ovulation data, PROC GLM in
SAS, version 8.0 (SAS Institute, Cary, NC) was used. The
model contained the following factors: treatment groups,
days treated, and their interaction. When a factor was statis-
tically significant, Bonferroni multiple comparison proce-
dure was used to adjust the level of significance.
RESULTS
Mice With hCG-Containing Osmotic Pump
Implants Exhibited Elevated Levels of Plasma
hCG and Developed Follicular Cysts Within 7
Days
To measure hCG levels, plasma was collected from hCG-
treated and sham-operated mice each day (n ϭ 8) for 7 days
after implantation surgery. Plasma concentrations of hCG
remained above 78 Ϯ 32 mIU/h during the 7-day period in
treated mice, whereas in sham-operated mice, hCG was
undetectable. To eliminate concern of hFSH trace contami-
nation in hCG infusion preparations, lots were tested for
hFSH. No detectable hFSH was found in either the hCG
preparation used to infuse animals or the plasma of hCG
pump-treated mice.
To assess the effect of chronic hCG treatment, we exam-
ined ovarian morphology (Fig. 1). Inspection of gross mor-
phology revealed abnormally enlarged, red ovaries with fol-
licular cysts in hCG osmotic pump-treated mice within 7
days as compared to sham-operated controls. The enlarge-
ment was bilateral and symmetrical. Ovaries were ovoid;
globular and cysts caused bulging of the surface. It should be
noted that hemorrhagic cysts were frequently observed in
these ovaries.
Analysis of tissues by H & E staining demonstrated
different sized follicles in all stages of maturation and atresia
with occasional corpora lutea. As is characteristic for cystic
follicles (3), cysts in this case had significantly fewer gran-
ulosa cells (compare Fig. 1A and B arrows). Although hy-
perplasia of the theca is sometimes observed in macrocystic
follicles such as these, we did not observe this in the 7-day
period of this study.
To quantify differences between sham-operated and hCG-
treated mice, ovarian wet weights of 10 ovaries from five
mice were measured. We also measured the perimeters of
follicles from 25 randomly selected follicles from sections of
10 ovaries from five mice (see Materials and Methods).
Ovaries from hCG-treated mice (9.7 Ϯ 3.3 mg) weighed
significantly (PϽ.05) more than ovaries from sham-operated
mice (2.1 Ϯ 0.4 mg). Similarly, perimeter measurements of
follicles in hCG-treated mice were significantly (PϽ.05)
larger (2,428 Ϯ 230 m) than those of sham-operated mice
(1,234 Ϯ 196 m). Thus, gross inspection, histological anal-
ysis, wet weight, and follicular size measurements revealed
the existence of follicular cysts in these mice.
Chronic Treatment With L-NAME During
Cystic Development Reduced Full Cystic
Development
When hCG-treated mice (hCG-only) were simultaneously
treated with L-NAME during cystic development (see Ma-
terials and Methods), follicular cysts were significantly
smaller in size by 18% Ϯ 9% (PϽ.05) Furthermore, these
smaller cysts had retained more granulosa cells than hCG-
only mice (compare Fig. 1B with D). Ovaries from mice
treated with hCG ϩ D-NAME had cystic follicles similar to
that of hCG-only treated animals (Fig. 1C).
Mice Treated Chronically With hCG
Exhibited High Levels of Plasma T That
Were Reduced to Control Levels by L-NAME
Because androgens in the ovary are produced in response
to LH/hCG signaling, we examined the effect of chronic
hCG treatment on T. By days 1 and 2, T levels were 0.5 Ϯ
FERTILITY & STERILITY 1303
4. 0.3 ng/mL, already significantly elevated over sham-oper-
ated controls (Ͻ0.05 ng/mL). Testosterone levels increased
significantly by day 3 (1.4 Ϯ 0.4 ng/mL) and stayed elevated
over the remaining 7-day period. Concentrations of T in
female rats treated chronically with hCG were significantly
elevated over sham-operated female animals and immature
males (both Ͻ0.05 ng/mL).
To test whether L-NAME could reduce T levels observed
in hCG-only mice, plasma T was measured in hCG ϩ
L-NAME mice. Because data from the mice treated only
with hCG showed that the most significant changes occurred
on days 2, 3, and 6, double pump experiments focused on
these time points. There was no significant difference at day
2 between the experimental groups (range, 0.27 Ϯ 0.15 to
0.58 Ϯ 0.19 ng/mL). At day 3, plasma T levels in sham and
NAME-treated mice were significantly lower (range, 0.23 Ϯ
0.08 to 0.35 Ϯ 0.18 ng/mL) than hCG-only mice (1.86 Ϯ
0.48 ng/mL). Similarly at day 6, plasma T levels in sham and
NAME-treated mice were still significantly lower (range,
0.27 Ϯ 0.18 to 0.78 Ϯ 0.29 ng/mL) than hCG-only mice
(1.46 Ϯ 0.24 ng/mL). When compared to each other, sham-
operated controls, hCG ϩ L-NAME-, and hCG ϩ
D-NAME-treated mice did not produce statistically different
results.
Ovulation Patterns in Mice Chronically
Exposed to hCG and L-NAME and D-NAME
To test BFB function and the ovulatory capacity of ova-
ries in hCG-treated mice, ovulation assays were performed
(Fig. 2). On indicated days after osmotic pump implantation,
animals were superovulated and COCs collected. Figure 2A
shows that on day 2, hCG-only mice ovulated significantly
fewer numbers of COCs (0.25 Ϯ 0.1 COCs) as compared to
sham-operated controls (40 Ϯ 13 COCs), but with continued
hCG infusion (days 3–7), hCG-only ovulated significantly
higher numbers of COCs (day 3: 13 Ϯ 5 COCs; day 7: 22 Ϯ
7 COCs) than their sham-operated counterparts (days 3 and
7: 5 Ϯ 2 COCs).
At day 2 in mice treated with hCG ϩ L-NAME and hCG
ϩ D-NAME (Fig. 2B), ovaries were not responsive to an
ovulatory surge in any of the experimental groups (range,
0.25 Ϯ 0.45 to 5.53 Ϯ 1.01 COCs) as compared to shams
(35.93 Ϯ 1.16 COCs). However, by days 3 and 6, treatment
groups yielded higher numbers of COCs than shams. Al-
though treatment with L-NAME yielded similar results on
both days 3 (21.2 Ϯ 4.31 COCs) and 6 (17.78 Ϯ 3.96
COCs), they were either statistically different from only
sham-operated mice (day 3) or sham-operated and hCG-only
treated mice (day 6). Similarly, treatment with D-NAME
yielded similar results on both days 3 (12.13 Ϯ 4.27 COCs)
and 6 (11.53 Ϯ 4.17 COCs), but when compared to other
groups were either not statistically different (day 3) or sta-
tistically different from all other groups (day 6).
F I G U R E 1
Effect of chronic hCG-only, hCG ϩ L-NAME, and hCG ϩ
D-NAME treatment on ovarian morphology. Immature preg-
nant mare serum gonadotropin (PMSG)-primed mice were
either sham-operated or implanted with osmotic pumps con-
taining either hCG or two pumps containing hCG and L-
NAME or hCG and D-NAME. (A), Section of an ovary from a
sham-operated mouse at day 7 after surgery (original mag-
nification, ϫ40) in which multiple layers of granulosa cells are
present. (B), A section of an ovary from an hCG-treated
mouse at day 7 after surgery (original magnification, ϫ40) in
which significantly fewer granulosa cells (arrows) are present
compared to ovary described in A. (C), Section of an ovary
from an hCG ϩ D-NAME mouse at day 7 after surgery
(original magnification, ϫ40), which is similar to A. (D), Sec-
tion of an ovary from an hCG ϩ L-NAME mouse at day 7 after
surgery (original magnification, ϫ40) in which more granulosa
cells (arrows) are present compared to ovary described in B.
F ϭ normal preovulatory follicle; CF ϭ cystic follicle.
Nemade. NO in murine ovarian follicular cysts. Fertil Steril 2002.
1304 Nemade et al. NO in murine ovarian follicular cysts Vol. 78, No. 6, December 2002
5. The BFB of Cystic Ovaries Did Not Respond
to Hormonal Stimuli Without NO
Immunohistochemical localization of the serum protein
I␣I has been previously demonstrated to serve as a marker
for the function of the BFB, as I␣I is excluded from the
interior of the preovulatory follicle until an hCG stimulus
(Fig. 3A and B) (8). Mice chronically treated with hCG were
given an ovulatory surge on indicated days and their BFB
examined. At day 3, I␣I influx indicated that the BFB of
follicles in treated ovaries was normal (Fig. 3C). However, at
days 4–7, cystic follicles were evident and the lack of I␣I
influx signified a closed and pathological BFB (Fig. 3D–F).
In Figure 3E and F, follicular cysts were adjacent to seem-
ingly normal follicles in which I␣I was present serving as
internal positive controls.
In previous studies, we have shown that normal function
of the BFB is dependent on the synthesis and release of NO
(9). Nitric oxide release causes hyperemia and edema of the
vasculature and thecal cells surrounding ovarian follicles
suggesting that NO plays a role in follicular development. To
test whether the dysfunction of the BFB in cystic follicles
was due to the loss of NO, we injected sodium nitroprusside
(2.5 mg/kg, IP) at day 7 in this mouse model and immuno-
localized I␣I. As shown in Figure 3G, I␣I presence inside the
follicle suggests that exogenous NO has the ability to open
the previously closed and hormonally unresponsive BFB
surrounding a cystic follicle.
The BFB in hCG and L-NAME
Simultaneously Treated Mice Was Sensitive to
an Ovulatory Surge
To test BFB function in mice simultaneously treated with
hCG and L-NAME and D-NAME, an ovulatory stimulus
was administered to 7-day treated mice. An ovulatory surge
in hCG ϩ D-NAME mice showed that I␣I was excluded
from cystic follicles (data not shown). When an ovulatory
surge was administered to hCG ϩ L-NAME-treated mice,
I␣I entered follicles signifying that the BFB was hormonally
responsive (compare Fig. 3H and I).
DISCUSSION
This study reports the involvement of NO in murine
ovarian follicular cysts. We have shown that chronic treat-
ment with hCG induces enlarged ovaries containing multiple
follicular cysts, which are approximately double the size of
follicles in sham-operated mice. These cysts enclose few, if
any granulosa cells, secrete high levels of T (as is expected
by the LH–thecal interstitial cell theory), and have impaired
ovarian BFB function. We have also shown that the inhibi-
tion of NOS by L-NAME during ovarian cystic formation
has the ability to reduce the size of follicular cysts, sustain
normal T levels, and maintain hormonal BFB reactivity in
cystic follicles.
F I G U R E 2
Ovulation patterns in hCG-only, hCG ϩ L-NAME, and hCG ϩ
D-NAME-treated mice on indicated days. (A), Cumulus oo-
cyte complexes were collected from sham-operated and
hCG-treated superovulated mice from days 2 through 7.
Sham-operated animals (n ϭ 15 for each day) are repre-
sented by open bars and hCG-treated mice are represented
by solid bars (n ϭ 30 for each day). Data represent the mean
Ϯ confidence interval; PϽ.05. *Statistically significant differ-
ence from the previous day within the same treatment group.
**Statistically significant difference between treatment
groups on a given day. (B), Cumulus oocyte complexes were
collected from superovulated mice on days 2, 3, and 6. Open
bars indicate sham-operated control animals (n ϭ 15). Solid
bars represent animals treated with hCG-only (n ϭ 30). Ver-
tical bars show data for animals treated with hCG ϩ L-NAME
(n ϭ 15). Horizontal bars show data for animals treated with
hCG ϩ D-NAME (n ϭ 10). Data represents the mean Ϯ
confidence interval.*PϽ.01 statistically different from all other
groups within a given time point. **PϽ.05 statistically differ-
ent from sham-operated mice. ***PϽ.05 statistically different
from sham-operated mice and hCG-only-treated mice.
Nemade. NO in murine ovarian follicular cysts. Fertil Steril 2002.
FERTILITY & STERILITY 1305
6. Ovulation assays revealed an unexpected time-dependent
increase in that exposure to constant hCG after day 2 pro-
duced high numbers of COCs instead of anovulation. We
hypothesize that the difference between sham-operated and
hCG-treated mice at day 2 is due to a delay of LH receptor
expression at the thecal and endothelial cell surface in a high
hCG milieu. The maintenance of hCG beyond day 2 up-
regulates and maintains levels of LH receptor making nor-
mal follicles continuously responsive to ovulatory surges.
Therefore, it is reasonable to hypothesize that abnormalities
in LH receptor expression or activity contribute to the
anovulation observed in follicular cyst disorders.
F I G U R E 3
Immunolocalization of inter-␣-inhibitor (I␣I) in follicles of mice treated with hCG-only, hCG ϩ L-NAME, and hCG ϩ D-NAME.
(A), Ovarian section from a sham-operated control mouse obtained 48 hours after pregnant mare serum gonadotropin (PMSG)
injection. Mice show I␣I present outside normal preovulatory follicles. (B), A positive control section from an ovary section
obtained 48 hours after PMSG and 6 hours after hCG localizes I␣I inside normal preovulatory follicles and around granulosa
cells. Chronic hCG-treated mice were treated with an ovulatory dose of hCG on indicated days, euthanized 6 hours later, and
ovaries were collected for I␣I analysis (C–F). (C), Section from an ovary with 3 days of chronic hCG treatment shows I␣I inside
follicles and around granulosa cells. (D), Section from an ovary with 4 days of chronic hCG treatment shows no signal in a cystic
follicle (arrow). (E), Section from an ovary with 6 days of chronic hCG treatment shows no signal in a cystic follicle (arrow), but
shows I␣I inside a normal follicle (arrow). (F), Section from an ovary with 7 days of chronic hCG treatment also shows no signal
in a cystic follicle (arrow), but shows I␣I inside a normal follicle (arrow). (G), Sodium nitroprusside was injected into 7-day
hCG-treated mice and euthanized 6 hours later. Inter-␣-inhibitor is present inside cystic follicles. (H), An ovary section from a
mouse treated with hCG ϩ L-NAME before an ovulatory surge shows mild to no I␣I signal inside follicles. (I), An ovary section
from a mouse treated with hCG ϩ L-NAME after an ovulatory surge shows I␣I inside follicles. F ϭ normal follicle; CF ϭ cystic
follicle. Bar ϭ 200 m.
Nemade. NO in murine ovarian follicular cysts. Fertil Steril 2002.
1306 Nemade et al. NO in murine ovarian follicular cysts Vol. 78, No. 6, December 2002
7. The impact of chronic hCG treatment on the BFB can be
visualized by immunolocalization of I␣I. The BFB opens in
response to an ovulatory surge and allows I␣I to enter
follicles and stabilize the hyaluronic acid cumulus extracel-
lular matrix (15, 16). Continuous hCG treatment caused
follicular cysts within 3 to 4 days. Upon an ovulatory surge,
immunohistochemical analysis revealed a lack of I␣I uptake
in cystic follicles indicating a defect in their BFB. However,
normal follicles within the same ovaries responded with
standard I␣I uptake. Therefore, we concluded that the BFB
is disrupted as a result of chronic levels of hCG/LH in
ovarian cyst disorders. As an explanation of why these mice
ovulated many COCs in the presence of cystic follicles with
dysfunctional blood follicle barriers, we speculate that COCs
collected at days 3–7 were ovulated from the population of
follicles that remained normal.
Treatment of mice with L-NAME during cystic develop-
ment resulted in follicular cysts that maintained hormonal
sensitivity at the level of the BFB, demonstrating that L-
NAME also had the ability to maintain vascular reactivity
observed in hCG-only treated mice. Presumably, in hCG-
only treated mice, NO is produced continually in response to
hCG infusion. In hCG ϩ L-NAME mice, inhibition of NO
production most likely reduces the effects of upstream hCG
stimulation. This situation allows these follicles to remain
sensitive to the endogenous NO generated from an ovulatory
surge. Follicles of mice treated with hCG ϩ D-NAME did
not contain I␣I, either before or after an ovulatory surge.
Thus, the inhibition of NOS, and thereby NO, moderates the
effects of hCG on the BFB, as well as cystic morphology.
On the basis of BFB responsiveness and T reduction in
mice treated with hCG ϩ L-NAME, it is clear that L-NAME
inhibits NOS at the level of the ovary, but it is possible that
an initial response to L-NAME is the up-regulation of NOS
protein activity leading to increased NO and ovulation ob-
served at day 3. Perhaps by day 6, decreased ovulation
results from inhibition at two levels: one is the chronic
hCG-induced down-regulation or desensitization of the LH
receptor and the other is the chronic inhibition of NOS
resulting in a reduction of NO and vasodilation. It is reason-
able to expect that the number of COCs ovulated from hCG
ϩ L-NAME-treated mice would mimic results from sham
controls. Surprisingly, these mice ovulated higher numbers
of COCs at days 3 and 6; thus, we hypothesize that because
these mice are still exposed to hCG, they experience some of
the effects of this hormone and are capable of ovulation.
Therefore, the ovulation patterns of hCG ϩ L-NAME mice
appear to be unique and warrant further investigation.
Although D-NAME was chosen to serve as a negative
control, in T measurements, D-NAME had similar effects to
L-NAME at days 2 and 3, and a slightly weaker NOS
inhibition at day 6. In ovulation assays, D-NAME simulta-
neous treatment exhibited either similar effects to L-NAME
(day 2), similar effects to hCG-only treated mice (day 3), or
unique effects (day 6). These studies confirm recent reports
that D-NAME has the ability to inhibit NOS (17, 18). The
mechanism by which D-NAME inhibits NOS is not yet
understood; however, once D-NAME enters an in vivo sys-
tem, any one of the following may occur to change its
biological activity: [1] D-NAME undergoes a D to L con-
version and thus, confers the inhibitory activity of L-NAME;
[2] the D-NAME molecule is fragmented yielding some
unknown metabolic NOS inhibitory byproduct; or [3] D-
NAME is demethylated and accepts some other molecule or
compound in place of the methyl group. In the present study,
we have shown that in a long-term experiment, D-NAME
provokes changes in ovarian physiology similar to L-NAME
and should be considered an NOS inhibitor.
Although these studies and others implicate NO in follic-
ular development, the investigation of its role in the vascu-
lature of follicular cyst formation is still in its infancy.
Because the few studies that exist on this topic focus on
humans, we will restrict our discussion to human subjects.
The most profound effect of NO is vasodilation, leading to
increased blood flow. With color Doppler ultrasonography,
researchers are now able to accurately localize vessels and
resolve blood flow velocity waveforms. During normal ovar-
ian development, rising levels of LH promote angiogenesis
and the ovary containing the dominant follicle is supplied
with the highest number of blood vessels providing the most
blood flow (19).
Thus, it is not surprising that several studies have docu-
mented increased ovarian stromal blood flow and vascular
endothelial growth factor (VEGF) in PCOS women (5, 13,
20). In PCOS patients, administration of the NO donor
glyceryl trinitrate increases vascular resistance of the uterine
artery of PCOS women suggesting that vascular responses
differ between PCOS women and normal women (21). High
blood flow may reflect a process of neoangiogenesis, forma-
tion of arteriovenous connections, or activity of vasoactive
compounds around the follicle and in the adjacent stroma
leading to cystic development.
Although the role of NO in follicular cysts is becoming
clearer, the therapeutic potential of this knowledge has yet to be
explored. Cystic ovarian disease and PCOS have necessitated a
wide range of therapies (22, 23). Typically the goal of live-
stock COD treatment is to regain fertility whereas, in humans,
the end point may be relief from hirsutism, abdominal men-
strual pain, hyperinsulinemia, or obesity in addition to infertility
treatment. Our current understanding of COD/PCOS only al-
lows for symptom-oriented treatment after the onset of the
disorder. There are few, if any, preventative treatments for
individuals predisposed to developing follicular cyst disorders
such as adolescents in whom irregular LH bursts and preco-
cious puberty are likely precursors of PCOS (24, 25). Thus,
results presented in this report provide the basis to explore
FERTILITY & STERILITY 1307
8. NOS inhibition as a potential preventative therapy for COD/
PCOS. Indeed, L-NAME is currently being tested for its
clinical use in septic shock (26, 27). As NOS inhibitors
become increasingly used in clinical treatments, studies inves-
tigating their effects on COD/PCOS will become inevitable.
In conclusion, our studies demonstrate that NO is involved
in the formation of hCG-induced follicular cysts and compli-
cations associated with follicular cyst disorders can be amelio-
rated by the NOS inhibitor, L-NAME. Administered during
cystic formation, L-NAME infusion reduced complete forma-
tion of cystic follicles, preserved BFB sensitivity, and reduced
T in these mice. We theorize that L-NAME reduces NO and
thereby blood flow to potentially cystic follicles and delays or
reduces pathogenesis. Further investigation will continue to
elucidate the role of NO in cystic follicles.
Acknowledgments: The authors thank Katryna Bogovich, Ph.D., University
of South Carolina, School of Medicine, Columbia, South Carolina, and
Anne N. Hirshfield, Ph.D., University of Maryland, School of Medicine,
Baltimore, Maryland, for their assistance with histological examination,
Leslie Myatt, Ph.D., University of Cincinnati, School of Medicine, Cincin-
nati, Ohio, for his guidance in the progress of this work, and Judy A. Bean,
Ph.D., M.P.H., Children’s Hospital Medical Center, Cincinnati, Ohio, for
her assistance with statistical analysis.
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1308 Nemade et al. NO in murine ovarian follicular cysts Vol. 78, No. 6, December 2002
