Hyperstimulation and a Gonadotropin-Releasing Hormone Agonist Modulate Ovarian Vascular Permeability by Altering Expression of the
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《内分泌学杂志》
Departments of Obstetrics and Gynecology (Y.K., T.E., K.N., K.M., H.H., T.B., T.H., T.S.) and Pathology (H.C., N.S.), Sapporo Medical University, School of Medicine, Sapporo 060-8556, Japan
Abstract
We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 μg/kg·d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm2) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.
Introduction
OVARIAN HYPERSTIMULATION SYNDROME (OHSS)is an important complication of ovulation induction for the treatment of infertility. It is characterized by massive extravascular fluid accumulation with hemoconcentration and cystic enlargement of the ovaries (1). The pathogenesis of OHSS has been investigated using a rat OHSS model developed by Ujioka et al. (2). Using that model, Gomez et al. (3) determined that hyperstimulation increases ovarian levels of the mRNAs encoding both vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2. They also showed that blocking VEGFR-2 reduced vascular permeability and suggested that this approach could be used to prevent OHSS in humans. Using the same OHSS model, we similarly observed increases in ovarian expression of VEGF, VEGFR-1, and VEGFR-2 mRNA and protein (4). In addition, we found that the levels of all three were significantly reduced by administration of a GnRH agonist (GnRHa). The ovaries of these hyperstimulated rats also showed increased vascular permeability, as indicated by increased Evans blue (EB) leakage, and that, too, was attenuated by GnRHa. Notably, those findings are consistent with our clinical observation that continuation of GnRHa administration for 1 wk after human chorionic gonadotropin (hCG) injection prevents early OHSS (5).
VEGF signaling is thought to mediate redistribution of tight junction proteins and the loss of the endothelial cell barrier architecture (6). Bearing that in mind, we have been working to better understand the molecular mechanism underlying OHSS by focusing on the expression of claudin-5, a 21- to 24-kDa endothelial cell-specific component of the tight junction strand that is also a key component of the blood-brain and blood-retina barriers (7, 8, 9, 10). We hypothesized that expression of claudin-5 is down-regulated when the ovaries are hyperstimulated in the rat OHSS model, which would in turn lead to increased vascular permeability as a result of altered barrier architecture, and that the beneficial effects of GnRHa would be reflected by an increase in the expression of claudin-5.
Materials and Methods
Hormones and drugs
Pregnant mare serum gonadotropin (PMSG) and hCG were obtained from Teikoku Hormone Manufactory Co. (Tokyo, Japan). The GnRHa analog leuprolide [(D-Leu6, des-Gly10 ethylamide) GnRH, a water-soluble dried peptide] was kindly provided by Takeda Chemical Industries, Ltd. (Osaka, Japan).
Animals
Immature female Sprague Dawley rats (25 d old, weighing 60–80 g) were obtained from Hokudo Co. (Sapporo, Japan) and were maintained in our laboratory on a 12-h light, 12-h dark regimen (lights on 0700–1900 h) with free access to water and a standard diet. The Animal Care and Use Committee of the Sapporo Medical University School of Medicine approved all procedures in this study, which were in accordance with the standards of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
The rats were randomly allocated to four groups: control rats were injected with 10 IU PMSG on d 27 of life, followed by injection of 10 IU hCG 48 h later on d 29. Hyperstimulated rats were injected with excessive doses of PMSG (50 IU/d) for 4 consecutive days (d 25 to 28), followed by 25 IU hCG on d 29. Short-term GnRHa-treated hyperstimulated rats received the same treatment as those in the hyperstimulated group but were also administered GnRHa (leuprolide 100 μg/kg·d in saline) twice a day (0900 and 1800 h) on d 29 and 30. And long-term GnRHa-treated hyperstimulated rats also received the same treatment as those in the hyperstimulated group but were administered GnRHa (twice a day) from d 25 to 30.
The following two series of experiments were performed 2 d after the hCG injections (d 31; 0900 h) and involved a total of 40 rats. In the first series, both ovaries from five rats in each of the four groups (n = 20) were weighed; the ovaries from one side were of each rat were then used for immunoblot analysis, and those from the other side were used for real-time PCR analysis. The blood was used for hormone assays. In the second series of experiments, one ovary was taken from five rats in each of the four groups (n = 20) and used to measure ovarian capillary permeability with EB; the other ovary was used for immunohistochemistry.
Evaluation of EB leakage
Beginning 2 d after hCG injection (d 31; 0900 h), rats from each of the four groups (n = 5 in each group) were injected via the inferior vena cava with 0.2 ml of 5 mM EB (Sigma, St. Louis, MO) under inhalation anesthesia with diethyl ether. The rats were then decapitated, and one ovary was removed and incubated in 2 ml of formamide for 24 h at 37 C. To evaluate the ovarian capillary permeability, the EB concentration in the formamide extract was measured as a function of light absorption at 620 nm determined using a spectrophotometer (BioSpec-1600; Shimadzu Corp., Kyoto, Japan). The ovarian EB content was expressed as nanograms per milligram tissue wet weight.
Hormone assays
Five rats from each group were decapitated 2 d after hCG injection, after which their blood was collected and serum estradiol and progesterone levels were determined using RIA kits from Diagnostic Products Corp. (Los Angeles, CA). The intra- and interassay coefficients of variation were 4.73 and 4.75%, respectively, for the estradiol RIA and 7.54 and 9.09%, respectively, for the progesterone RIA.
Immunohistochemistry
For immunohistochemical detection of claudin-5 and von Willebrand factor (vWF), ovaries were fixed for 12 h at 4 C in 4% buffered formalin and then routinely processed for paraffin embedding at 56 C, after which serial sections (5 μm) were cut and stretched at 45 C, allowed to dry, and stored at 4 C until use. Immunohistochemistry was carried out using the avidin-biotin-peroxidase complex technique with a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, the slides were deparaffinized and then incubated for 30 min at room temperature, first with 0.6% H2O2 in methanol to block endogenous peroxidase activity and then with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan) to minimize nonspecific staining. After rinsing several times with PBS, the slides were incubated for 60 min at room temperature with mouse monoclonal antimouse claudin-5 (Zymed, San Francisco, CA) or rabbit polyclonal antihuman vWF (DakoCytomation, Kyoto, Japan) and then with biotinylated goat antimouse or antirabbit IgG and avidin-biotin-peroxidase complex. Antigen-antibody complexes were visualized using diaminobenzidine as a chromogen. The sections were lightly counterstained with Meyer’s hematoxylin. As a control, normal rabbit serum was used or the primary antibody was omitted (data not shown).
Quantification of microvessels
Vessel densities were quantified as described as described previously (11). Histologic slides were blind coded during the assessment and viewed at x100 magnification (x10 objective lens and x10 ocular lens; 0.581 mm2/field). Tissue images were captured with a digital camera (Olympus, Inc., Tokyo, Japan). For each section, at least 10 randomly selected fields were counted to determine the average vessel density and assess the heterogeneity of vessel densities within the ovaries. The total tissue area analyzed in each section was 5.81 mm2.
The area and number of vWF-positive vessels were quantified using the software ImagePro Plus (version 5.0; Media Cybernetics, Silver Spring, MD). The vessel endothelial area (percent of the total) in each field was calculated as: (area of vWF-positive endothelium/measured tissue area) x 100%. Vessel density (number/10 mm2) in each field was calculated as the number of vWF-positive vessels/0.581 mm2. Large-vessel density (density of vessels > 30 μm in diameter) was also measured. Mean values in each group were calculated from five samples.
Immunoblot analysis
The proteins in samples of ovarian extract (100 μg protein) were separated using a commercial polyacrylamide gel (REDYGELS J; Bio-Rad Laboratories, Hercules, CA) and transferred to polyvinyl difluoride membranes (Immobilon-P; Millipore, Billerica, MA) using an electroblotting apparatus. The membranes were then blocked overnight at room temperature in 5% wt/vol nonfat milk in PBS containing 0.05% Tween 20 (TPBS) on an orbital shaker and washed five times for 5 min each in TPBS. Thereafter the membranes were incubated with antimouse claudin-5 antibody (1 μg/ml) for 2 h at room temperature in a humidified chamber, washed five times for 5 min each in TPBS, and then incubated for 1 h with horseradish peroxidase-conjugated antirabbit IgG (DACO, Glostrup, Denmark). After washing again five times for 6 min each in TPBS, the membranes were incubated with Amersham enhanced chemiluminescence reagent for 5 min and exposed to x-ray film (Hyper-film ECL, Amersham, Buckinghamshire, UK) for several minutes in a dark room. Relative levels of protein expression were then determined by densitometry using National Institutes of Health Image. A mouse monoclonal anti--actin antibody (Sigma) served as an internal control.
Real-time RT-PCR
Total RNA from samples of ovarian extract was reverse transcribed before real-time PCR amplification as described previously (12). Briefly, PCR was carried out using a DyNAmo HS SYBR Green qPCR kit (Finnzymes, Espoo, Finland), and the fluorescent signals were detected using an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). The nucleotide sequences of the primers used were: rat claudin-5 (accession no. NM_031701), 5'-cgcttgtggcactctttgt-3' (forward) and 5'-actcccggactacgatgttg-3' (reverse); and rat -actin (accession no. NM_031144), 5'-gtcgacaacggctccggca-3' (forward) and 5'-gtcaggtcccggccagcca-3' (reverse). The thermal cycling protocol entailed an initial denaturation step at 95 C for 15 min and 40 cycles at 94 C for 10 sec, 60 C for 20 sec, and 72 C for 30 sec. Experiments were carried out in triplicate, and the levels of claudin-5 mRNA were normalized to those of rat -actin. The ratio obtained in the control group was assigned a value of 1.
Data analysis
Data are presented as means ± SE. Differences between the groups were evaluated using one-way ANOVA and Student-Neuman-Keuls test. Values of P < 0.05 were considered significant.
Results
Measurement of ovary weight, serum hormone levels, and ovarian capillary permeability
The effects of GnRHa on ovary weights, serum estradiol and progesterone, and ovarian capillary permeability (EB leakage) are summarized in Table 1. Ovary weights in hyperstimulated rats were about four times greater than in control rats, whereas serum estradiol and progesterone levels were, respectively, about 10 and 13 times higher in hyperstimulated rats than control rats. Likewise, ovarian EB content was significantly higher in hyperstimulated rats than control rats, indicating a significant increase in vascular permeability. All of these effects of hyperstimulation were reversed by GnRHa administration. Indeed, long-term GnRHa tended to reduce serum hormone concentrations to levels below control, although the difference from control was not significant.
Effect of GnRHa on expression of claudin-5
Immunohistochemical staining revealed claudin-5 protein to be localized to the endothelial cells within the ovaries; moreover, the staining was stronger in the control and long-term GnRHa groups than the hyperstimulated and short-term GnRHa groups, suggesting greater expression of claudin-5 in the first two (Fig. 1). No vessel greater than 30 μm in diameter stained for cluadin-5, and no staining was seen in the negative controls (data not shown).
Consistent with the immunohistochemistry, we detected 21-kDa claudin-5 protein as well as its mRNA in ovarian extracts (Figs. 2 and 3). Compared with control, hyperstimulation significantly reduced expression of both the mRNA and protein by about 50%, but the inhibitory effect of hyperstimulation was completely reversed by long-term GnRHa treatment (Figs. 2B and 3).
Effect of GnRHa on ovarian vascular density
Accompanying the hyperstimulation-induced down-regulation of claudin-5 expression was a significant increase in vessel endothelial area (Fig. 2A). This increase was not seen in hyperstimulated rats treated with GnRHa, indicating that both short- and long-term GnRHa treatment significantly reduced endothelial area. On the other hand, microvessel numbers were significantly lower in control rats than the other three groups, indicating GnRHa had no significant effect on capillary density (Fig. 4B). But also increased in hyperstimulated rats was the density of vessels greater than 30 μm in diameter, and this increase was significantly attenuated by long-term GnRHa treatment, which likely accounts for the GnRHa-induced decline in endothelial area (Fig. 4C).
Discussion
OHSS is characterized by increased vascular permeability, ovarian enlargement, and, in the most severe cases, significant morbidity and even mortality (13). Putative factors involved in the pathogenesis of OHSS include VEGF (3, 4, 14), the kinin-kallikrein system (15, 16), the renin-angiotensin system (17), and various cytokines (18, 19); however, the specific molecular mechanism for the increased ovarian vascular permeability has remained unclear. In the present study, we evaluated ovarian expression of the endothelium-specific tight junction protein claudin-5 in a rat OHSS model and the effects of treatment with GnRHa. We found that hyperstimulation significantly reduces ovarian levels of both claudin-5 protein and its mRNA and significantly elevates serum estradiol and progesterone levels and ovarian capillary development and vascular permeability. Histologically, fewer claudin-5-positive vessels were detected than vWF-positive vessels, which is consistent with the earlier finding that claudin-5 is present in capillaries and arteriolar endothelial cells but not in venules or veins (20). It is noteworthy that microvessel density in hyperstimulated rats was unaffected by either short- or long-term GnRHa treatment but that long-term GnRHa treatment significantly reduced the density of vessels greater than 30 μm in diameter. This suggests that the increase in permeability seen in hyperstimulated rats is related to vessel size and not vessel number.
GnRHa was used in the present study to inhibit gonadotropic effects on the ovary. There is increasing evidence of extrapituitary effects of GnRH on the gonads of both humans and rats (21, 22). Indeed, it has been shown that human ovary and granulose-luteal cells express mRNAs for both GnRH and its receptor (23, 24). Functionally, moreover, GnRH reportedly regulates steroidogenesis (25, 26), the MAPK cascade (27), and apoptosis (28) and inhibits FSH-induced cAMP-dependent responses (29) in human granulose-luteal cells. And in rat a variety of functions has been suggested for ovarian GnRH, including a role in oocyte maturation (30) and follicular atresia or selection (31), an effect on the corpus luteum (32), and an effect on the fertilization process (33).
Levels of VEGF mRNA are reportedly elevated in hyperstimulated rats (4, 5). In addition, Wang et al. (34) reported that VEGF increases vascular permeability by reducing tight junction occludin expression and disrupting ZO-1 and occludin organization, leading to tight junction disassembly. Consistent with those findings, Levin et al. (35) demonstrated that VEGF is the follicular fluid factor responsible for increased endothelial cell permeability in OHSS and suggested its presence could serve as a marker of the syndrome. Rearrangement of the actin cytoskeleton and disruption of ZO-1 protein are likely primary mechanisms by which VEGF in follicular fluid disrupts the endothelial cell tight junctions and increases permeability. Our findings indicate that down-regulation of claudin-5 expression is also a key contributor to the increased ovarian vascular permeability seen in OHSS.
That serum estradiol levels were elevated in hyperstimulated rats also might contribute to the increased vascular permeability. Evidence suggests that endothelial cells express estrogen receptors (36) and that estrogen modulates paracellular permeability of human endothelial cells via endothelial nitric oxide synthase- and inducible nitric oxide synthase-related mechanisms (37). GnRHa inhibits steroidogenic responses to hormonal stimulation and blocks luteinization in superovulated rats (38, 39, 40), suggesting that the GnRHa-induced decline in estradiol levels seen in the present study contributed to the accompanying reduction in vascular permeability.
In conclusion, our findings indicate that changes in the expression of the tight junction protein claudin-5 significantly affect ovarian vascular permeability in an OHSS rat model. This suggests that preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture may be the key to protecting patients against massive extravascular fluid accumulation in OHSS.
Acknowledgments
We are grateful to Dr. H. R. Behrman (Department of Obstetrics/Gynecology, Yale Medical School, New Haven, CT) for helpful discussion and advice.
Footnotes
This work was supported by Grant-Aid for Scientific Research 14571574 from the Japanese Ministry of Education, Science, and Culture.
First Published Online November 3, 2005
Abbreviations: EB, Evans blue; GnRHa, GnRH agonist; hCG, human chorionic gonadotropin; OHSS, ovarian hyperstimulation syndrome; PMSG, pregnant mare serum gonadotropin; TPBS, PBS containing Tween 20; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; vWF, von Willebrand factor.
Accepted for publication October 24, 2005.
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Abstract
We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 μg/kg·d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm2) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.
Introduction
OVARIAN HYPERSTIMULATION SYNDROME (OHSS)is an important complication of ovulation induction for the treatment of infertility. It is characterized by massive extravascular fluid accumulation with hemoconcentration and cystic enlargement of the ovaries (1). The pathogenesis of OHSS has been investigated using a rat OHSS model developed by Ujioka et al. (2). Using that model, Gomez et al. (3) determined that hyperstimulation increases ovarian levels of the mRNAs encoding both vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2. They also showed that blocking VEGFR-2 reduced vascular permeability and suggested that this approach could be used to prevent OHSS in humans. Using the same OHSS model, we similarly observed increases in ovarian expression of VEGF, VEGFR-1, and VEGFR-2 mRNA and protein (4). In addition, we found that the levels of all three were significantly reduced by administration of a GnRH agonist (GnRHa). The ovaries of these hyperstimulated rats also showed increased vascular permeability, as indicated by increased Evans blue (EB) leakage, and that, too, was attenuated by GnRHa. Notably, those findings are consistent with our clinical observation that continuation of GnRHa administration for 1 wk after human chorionic gonadotropin (hCG) injection prevents early OHSS (5).
VEGF signaling is thought to mediate redistribution of tight junction proteins and the loss of the endothelial cell barrier architecture (6). Bearing that in mind, we have been working to better understand the molecular mechanism underlying OHSS by focusing on the expression of claudin-5, a 21- to 24-kDa endothelial cell-specific component of the tight junction strand that is also a key component of the blood-brain and blood-retina barriers (7, 8, 9, 10). We hypothesized that expression of claudin-5 is down-regulated when the ovaries are hyperstimulated in the rat OHSS model, which would in turn lead to increased vascular permeability as a result of altered barrier architecture, and that the beneficial effects of GnRHa would be reflected by an increase in the expression of claudin-5.
Materials and Methods
Hormones and drugs
Pregnant mare serum gonadotropin (PMSG) and hCG were obtained from Teikoku Hormone Manufactory Co. (Tokyo, Japan). The GnRHa analog leuprolide [(D-Leu6, des-Gly10 ethylamide) GnRH, a water-soluble dried peptide] was kindly provided by Takeda Chemical Industries, Ltd. (Osaka, Japan).
Animals
Immature female Sprague Dawley rats (25 d old, weighing 60–80 g) were obtained from Hokudo Co. (Sapporo, Japan) and were maintained in our laboratory on a 12-h light, 12-h dark regimen (lights on 0700–1900 h) with free access to water and a standard diet. The Animal Care and Use Committee of the Sapporo Medical University School of Medicine approved all procedures in this study, which were in accordance with the standards of the National Institutes of Health Guide for Care and Use of Laboratory Animals.
The rats were randomly allocated to four groups: control rats were injected with 10 IU PMSG on d 27 of life, followed by injection of 10 IU hCG 48 h later on d 29. Hyperstimulated rats were injected with excessive doses of PMSG (50 IU/d) for 4 consecutive days (d 25 to 28), followed by 25 IU hCG on d 29. Short-term GnRHa-treated hyperstimulated rats received the same treatment as those in the hyperstimulated group but were also administered GnRHa (leuprolide 100 μg/kg·d in saline) twice a day (0900 and 1800 h) on d 29 and 30. And long-term GnRHa-treated hyperstimulated rats also received the same treatment as those in the hyperstimulated group but were administered GnRHa (twice a day) from d 25 to 30.
The following two series of experiments were performed 2 d after the hCG injections (d 31; 0900 h) and involved a total of 40 rats. In the first series, both ovaries from five rats in each of the four groups (n = 20) were weighed; the ovaries from one side were of each rat were then used for immunoblot analysis, and those from the other side were used for real-time PCR analysis. The blood was used for hormone assays. In the second series of experiments, one ovary was taken from five rats in each of the four groups (n = 20) and used to measure ovarian capillary permeability with EB; the other ovary was used for immunohistochemistry.
Evaluation of EB leakage
Beginning 2 d after hCG injection (d 31; 0900 h), rats from each of the four groups (n = 5 in each group) were injected via the inferior vena cava with 0.2 ml of 5 mM EB (Sigma, St. Louis, MO) under inhalation anesthesia with diethyl ether. The rats were then decapitated, and one ovary was removed and incubated in 2 ml of formamide for 24 h at 37 C. To evaluate the ovarian capillary permeability, the EB concentration in the formamide extract was measured as a function of light absorption at 620 nm determined using a spectrophotometer (BioSpec-1600; Shimadzu Corp., Kyoto, Japan). The ovarian EB content was expressed as nanograms per milligram tissue wet weight.
Hormone assays
Five rats from each group were decapitated 2 d after hCG injection, after which their blood was collected and serum estradiol and progesterone levels were determined using RIA kits from Diagnostic Products Corp. (Los Angeles, CA). The intra- and interassay coefficients of variation were 4.73 and 4.75%, respectively, for the estradiol RIA and 7.54 and 9.09%, respectively, for the progesterone RIA.
Immunohistochemistry
For immunohistochemical detection of claudin-5 and von Willebrand factor (vWF), ovaries were fixed for 12 h at 4 C in 4% buffered formalin and then routinely processed for paraffin embedding at 56 C, after which serial sections (5 μm) were cut and stretched at 45 C, allowed to dry, and stored at 4 C until use. Immunohistochemistry was carried out using the avidin-biotin-peroxidase complex technique with a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, the slides were deparaffinized and then incubated for 30 min at room temperature, first with 0.6% H2O2 in methanol to block endogenous peroxidase activity and then with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan) to minimize nonspecific staining. After rinsing several times with PBS, the slides were incubated for 60 min at room temperature with mouse monoclonal antimouse claudin-5 (Zymed, San Francisco, CA) or rabbit polyclonal antihuman vWF (DakoCytomation, Kyoto, Japan) and then with biotinylated goat antimouse or antirabbit IgG and avidin-biotin-peroxidase complex. Antigen-antibody complexes were visualized using diaminobenzidine as a chromogen. The sections were lightly counterstained with Meyer’s hematoxylin. As a control, normal rabbit serum was used or the primary antibody was omitted (data not shown).
Quantification of microvessels
Vessel densities were quantified as described as described previously (11). Histologic slides were blind coded during the assessment and viewed at x100 magnification (x10 objective lens and x10 ocular lens; 0.581 mm2/field). Tissue images were captured with a digital camera (Olympus, Inc., Tokyo, Japan). For each section, at least 10 randomly selected fields were counted to determine the average vessel density and assess the heterogeneity of vessel densities within the ovaries. The total tissue area analyzed in each section was 5.81 mm2.
The area and number of vWF-positive vessels were quantified using the software ImagePro Plus (version 5.0; Media Cybernetics, Silver Spring, MD). The vessel endothelial area (percent of the total) in each field was calculated as: (area of vWF-positive endothelium/measured tissue area) x 100%. Vessel density (number/10 mm2) in each field was calculated as the number of vWF-positive vessels/0.581 mm2. Large-vessel density (density of vessels > 30 μm in diameter) was also measured. Mean values in each group were calculated from five samples.
Immunoblot analysis
The proteins in samples of ovarian extract (100 μg protein) were separated using a commercial polyacrylamide gel (REDYGELS J; Bio-Rad Laboratories, Hercules, CA) and transferred to polyvinyl difluoride membranes (Immobilon-P; Millipore, Billerica, MA) using an electroblotting apparatus. The membranes were then blocked overnight at room temperature in 5% wt/vol nonfat milk in PBS containing 0.05% Tween 20 (TPBS) on an orbital shaker and washed five times for 5 min each in TPBS. Thereafter the membranes were incubated with antimouse claudin-5 antibody (1 μg/ml) for 2 h at room temperature in a humidified chamber, washed five times for 5 min each in TPBS, and then incubated for 1 h with horseradish peroxidase-conjugated antirabbit IgG (DACO, Glostrup, Denmark). After washing again five times for 6 min each in TPBS, the membranes were incubated with Amersham enhanced chemiluminescence reagent for 5 min and exposed to x-ray film (Hyper-film ECL, Amersham, Buckinghamshire, UK) for several minutes in a dark room. Relative levels of protein expression were then determined by densitometry using National Institutes of Health Image. A mouse monoclonal anti--actin antibody (Sigma) served as an internal control.
Real-time RT-PCR
Total RNA from samples of ovarian extract was reverse transcribed before real-time PCR amplification as described previously (12). Briefly, PCR was carried out using a DyNAmo HS SYBR Green qPCR kit (Finnzymes, Espoo, Finland), and the fluorescent signals were detected using an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). The nucleotide sequences of the primers used were: rat claudin-5 (accession no. NM_031701), 5'-cgcttgtggcactctttgt-3' (forward) and 5'-actcccggactacgatgttg-3' (reverse); and rat -actin (accession no. NM_031144), 5'-gtcgacaacggctccggca-3' (forward) and 5'-gtcaggtcccggccagcca-3' (reverse). The thermal cycling protocol entailed an initial denaturation step at 95 C for 15 min and 40 cycles at 94 C for 10 sec, 60 C for 20 sec, and 72 C for 30 sec. Experiments were carried out in triplicate, and the levels of claudin-5 mRNA were normalized to those of rat -actin. The ratio obtained in the control group was assigned a value of 1.
Data analysis
Data are presented as means ± SE. Differences between the groups were evaluated using one-way ANOVA and Student-Neuman-Keuls test. Values of P < 0.05 were considered significant.
Results
Measurement of ovary weight, serum hormone levels, and ovarian capillary permeability
The effects of GnRHa on ovary weights, serum estradiol and progesterone, and ovarian capillary permeability (EB leakage) are summarized in Table 1. Ovary weights in hyperstimulated rats were about four times greater than in control rats, whereas serum estradiol and progesterone levels were, respectively, about 10 and 13 times higher in hyperstimulated rats than control rats. Likewise, ovarian EB content was significantly higher in hyperstimulated rats than control rats, indicating a significant increase in vascular permeability. All of these effects of hyperstimulation were reversed by GnRHa administration. Indeed, long-term GnRHa tended to reduce serum hormone concentrations to levels below control, although the difference from control was not significant.
Effect of GnRHa on expression of claudin-5
Immunohistochemical staining revealed claudin-5 protein to be localized to the endothelial cells within the ovaries; moreover, the staining was stronger in the control and long-term GnRHa groups than the hyperstimulated and short-term GnRHa groups, suggesting greater expression of claudin-5 in the first two (Fig. 1). No vessel greater than 30 μm in diameter stained for cluadin-5, and no staining was seen in the negative controls (data not shown).
Consistent with the immunohistochemistry, we detected 21-kDa claudin-5 protein as well as its mRNA in ovarian extracts (Figs. 2 and 3). Compared with control, hyperstimulation significantly reduced expression of both the mRNA and protein by about 50%, but the inhibitory effect of hyperstimulation was completely reversed by long-term GnRHa treatment (Figs. 2B and 3).
Effect of GnRHa on ovarian vascular density
Accompanying the hyperstimulation-induced down-regulation of claudin-5 expression was a significant increase in vessel endothelial area (Fig. 2A). This increase was not seen in hyperstimulated rats treated with GnRHa, indicating that both short- and long-term GnRHa treatment significantly reduced endothelial area. On the other hand, microvessel numbers were significantly lower in control rats than the other three groups, indicating GnRHa had no significant effect on capillary density (Fig. 4B). But also increased in hyperstimulated rats was the density of vessels greater than 30 μm in diameter, and this increase was significantly attenuated by long-term GnRHa treatment, which likely accounts for the GnRHa-induced decline in endothelial area (Fig. 4C).
Discussion
OHSS is characterized by increased vascular permeability, ovarian enlargement, and, in the most severe cases, significant morbidity and even mortality (13). Putative factors involved in the pathogenesis of OHSS include VEGF (3, 4, 14), the kinin-kallikrein system (15, 16), the renin-angiotensin system (17), and various cytokines (18, 19); however, the specific molecular mechanism for the increased ovarian vascular permeability has remained unclear. In the present study, we evaluated ovarian expression of the endothelium-specific tight junction protein claudin-5 in a rat OHSS model and the effects of treatment with GnRHa. We found that hyperstimulation significantly reduces ovarian levels of both claudin-5 protein and its mRNA and significantly elevates serum estradiol and progesterone levels and ovarian capillary development and vascular permeability. Histologically, fewer claudin-5-positive vessels were detected than vWF-positive vessels, which is consistent with the earlier finding that claudin-5 is present in capillaries and arteriolar endothelial cells but not in venules or veins (20). It is noteworthy that microvessel density in hyperstimulated rats was unaffected by either short- or long-term GnRHa treatment but that long-term GnRHa treatment significantly reduced the density of vessels greater than 30 μm in diameter. This suggests that the increase in permeability seen in hyperstimulated rats is related to vessel size and not vessel number.
GnRHa was used in the present study to inhibit gonadotropic effects on the ovary. There is increasing evidence of extrapituitary effects of GnRH on the gonads of both humans and rats (21, 22). Indeed, it has been shown that human ovary and granulose-luteal cells express mRNAs for both GnRH and its receptor (23, 24). Functionally, moreover, GnRH reportedly regulates steroidogenesis (25, 26), the MAPK cascade (27), and apoptosis (28) and inhibits FSH-induced cAMP-dependent responses (29) in human granulose-luteal cells. And in rat a variety of functions has been suggested for ovarian GnRH, including a role in oocyte maturation (30) and follicular atresia or selection (31), an effect on the corpus luteum (32), and an effect on the fertilization process (33).
Levels of VEGF mRNA are reportedly elevated in hyperstimulated rats (4, 5). In addition, Wang et al. (34) reported that VEGF increases vascular permeability by reducing tight junction occludin expression and disrupting ZO-1 and occludin organization, leading to tight junction disassembly. Consistent with those findings, Levin et al. (35) demonstrated that VEGF is the follicular fluid factor responsible for increased endothelial cell permeability in OHSS and suggested its presence could serve as a marker of the syndrome. Rearrangement of the actin cytoskeleton and disruption of ZO-1 protein are likely primary mechanisms by which VEGF in follicular fluid disrupts the endothelial cell tight junctions and increases permeability. Our findings indicate that down-regulation of claudin-5 expression is also a key contributor to the increased ovarian vascular permeability seen in OHSS.
That serum estradiol levels were elevated in hyperstimulated rats also might contribute to the increased vascular permeability. Evidence suggests that endothelial cells express estrogen receptors (36) and that estrogen modulates paracellular permeability of human endothelial cells via endothelial nitric oxide synthase- and inducible nitric oxide synthase-related mechanisms (37). GnRHa inhibits steroidogenic responses to hormonal stimulation and blocks luteinization in superovulated rats (38, 39, 40), suggesting that the GnRHa-induced decline in estradiol levels seen in the present study contributed to the accompanying reduction in vascular permeability.
In conclusion, our findings indicate that changes in the expression of the tight junction protein claudin-5 significantly affect ovarian vascular permeability in an OHSS rat model. This suggests that preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture may be the key to protecting patients against massive extravascular fluid accumulation in OHSS.
Acknowledgments
We are grateful to Dr. H. R. Behrman (Department of Obstetrics/Gynecology, Yale Medical School, New Haven, CT) for helpful discussion and advice.
Footnotes
This work was supported by Grant-Aid for Scientific Research 14571574 from the Japanese Ministry of Education, Science, and Culture.
First Published Online November 3, 2005
Abbreviations: EB, Evans blue; GnRHa, GnRH agonist; hCG, human chorionic gonadotropin; OHSS, ovarian hyperstimulation syndrome; PMSG, pregnant mare serum gonadotropin; TPBS, PBS containing Tween 20; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; vWF, von Willebrand factor.
Accepted for publication October 24, 2005.
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