Ovarian and peripheral blood inhibin concentrations increase with gonadotropin treatment in immature rats

The effects of PMSG treatment on ovarian and circulating inhibin concentrations in immature female rats has been examined. Sixty-four hours after injection of 10, 20 or 40 IU of PMSG the animals were anesthetized with ether; ovaries, uteri and blood from the abdominal aorta were collected. Steroid-free extracts of ovary and serum samples were prepared and assayed quantitatively for inhibin activity in an in vitro bioassay system. PMSG treatment elevated (p less than 0.001) both uterine and ovarian wt, and ovarian and peripheral concentrations of inhibin. A dose-related increase occurred ovarian wt, and in peripheral and ovarian content of inhibin. Ovarian inhibin concentration increased with dose of PMSG until the highest dose, where a significant decline and luteinization were seen. Peripheral FSH levels were significantly lowered at all doses of PMSG treatment; in contrasts, LH was significantly elevated, due to cross-reaction of PMSG in the LH assay. These results show that both ovarian and circulating levels of inhibin are related to the degree of gonadotropic stimulation, supporting the view that inhibin is involved in folliculogenesis and in the feedback regulation of FSH.     

Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization

GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.     

Alterations in follicle development, steroidogenesis, and gonadotropin receptor binding in a model of ovulatory blockade

Immature female rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before gonadotropin-induced follicle development and ovulation ovulate significantly fewer ova compared with controls. This study was designed to investigate potential ovarian-specific mechanisms of TCDD-mediated inhibition of ovulation. Immature hypophysectomized rats were treated with TCDD (32 microg/kg) or corn oil vehicle. Follicle development was initiated by injection of 10 IU PMSG 24 h after TCDD, and ovulation was induced 52 h after PMSG by injection of 10 IU hCG. The number of ova flushed from the oviduct was assessed the morning after hCG injection, and ovaries were collected at multiple times throughout the treatment schedule for histological analysis and analysis of FSH and hCG receptor binding and ovarian cAMP levels. In addition, serum levels of estradiol and progesterone were determined. Control rats ovulated 9.3 +/- 1.5 ova, whereas TCDD-treated rats ovulated 0.6 +/- 0.3. Gonadotropin receptor binding was evaluated 52 h after PMSG at the usual time of hCG injection to induce ovulation. Both FSH binding and hCG binding were significantly reduced in animals treated with TCDD. Serum estradiol levels in control animals were increased by 52 h after PMSG administration. In contrast, serum levels of estradiol in TCDD-treated animals remained low. In response to the ovulatory dose of hCG, serum levels of both estradiol and progesterone increased in control animals. Steroid levels also increased in TCDD-treated animals, but did not reach the peak levels observed in controls. TCDD treatment further resulted in lower ovarian cAMP levels at 52 h post-PMSG and at 5 h post-hCG. Together the data indicate that TCDD treatment altered the ability of the ovary to respond to PMSG, resulting in the development of follicles not comparable to controls (lower gonadotropin binding, lower estradiol production, lower levels of cAMP). It appears that critical steps in the development and maturation of follicles are disrupted by TCDD.     

Human chorionic gonadotrophin-induced suppression of serum inhibin involves reduction in ovarian inhibin alpha-subunit messenger RNA levels in the pregnant mare serum gonadotrophin-primed female rat

We have investigated the effect of administration of human chorionic gonadotrophin (hCG) to immature female rats pretreated with pregnant mare serum gonadotrophin (PMSG) on ovarian inhibin alpha-subunit mRNA, serum inhibin and circulating gonadotrophin levels. PMSG stimulation alone caused a 5-fold increase in relative inhibin alpha-subunit mRNA levels and a 12-fold increase in serum inhibin by 48 h. However, injection of hCG at 40 h suppressed PMSG-stimulated ovarian inhibin alpha-subunit mRNA and serum inhibin to levels at 48 h not statistically significantly different from controls. Serum follicle-stimulating hormone (FSH) fell after PMSG treatment, but rose after combined PMSG-hCG treatment. Serum luteinizing hormone (LH) was unchanged after PMSG alone but also rose after combined PMSG and hCG therapy. In conclusion, hCG suppresses ovarian inhibin synthesis in PMSG-stimulated immature female rats with preservation of the reciprocal relationship between inhibin and FSH. This immature female rat model therefore provides further insight into the effects of LH or hCG on granulosa cell inhibin production just prior to ovulation.     

Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.     

Hormonal regulation of proprotein convertase subtilisin/kexin type 5 expression during ovarian follicle development in the rat

The proprotein convertase subtilisin/kexin (PCSKs), a family of subtilisin-like proteases, is the processing enzymes for the activation of many hormone precursors. The present study was designed to identify the PCSK isoform expressed in the ovary and to examine its expression in gonadotropin-stimulated rat ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of Pcsk5 messenger RNA (mRNA) during development with the highest levels at 21 days of age. Treatment of immature rats with PMSG further increased ovarian Pcsk5 expression, and in situ hybridization analysis revealed the localization of Pcsk5 mRNA in theca-interstitial cells of follicles in different sizes. Interestingly, treatment of PMSG-primed rats with hCG resulted in a transient stimulation of ovarian Pcsk5 mRNA levels within 3-6 h. In addition to theca-interstitial cells, hCG treatment induced the expression of Pcsk5 in granulosa cells of preovulatory follicles. Pcsk1, 2 and 4 mRNAs were not detected whereas Pcsk7 mRNA was slightly expressed. Injection of a progestin antagonist RU486 or an inhibitor of 3beta-hydroxysteroid dehydrogenase epostane at 1h before hCG treatment inhibited hCG-induced Pcsk5 mRNA levels. Treatment with LH stimulated both Pcsk5 mRNA and protein levels in preovulatory follicles cultured in vitro. In addition, forskolin but not TPA stimulated Pcsk5 mRNA levels. RNase protection assay revealed that the soluble Pcsk5A variant was the predominant form stimulated by gonadotropins in the ovary. Finally, the predicted proprotein substrates cleaved by PCSK5 were analyzed in preovulatory follicles using regular expressions. The present study demonstrates PCSK5A as the gonadotropin-regulated PCSK isoform in the ovary, and its possible contribution to ovulation by processing pro-TGFbeta and matrix metalloproteinase family.     

Increase in ovarian 15-hydroxyeicosatetraenoic acid during ovulation in the gonadotropin-primed immature rat

The ovarian level of 15-hydroxyeicosatetraenoic acid (15-HETE) was measured by RIA during ovulation in gonadotropin-primed immature Wistar rats. The ovulatory process was initiated in 25-day-old rats by a 10-IU injection of hCG sc 2 days after the animals had been primed with 10 IU PMSG, sc. Ovarian follicles begin to ovulate 10 h after hCG. At 0 h after hCG, the ovarian 15-HETE level was 0.6 +/- 0.2 ng/mg ovarian protein. At 6 h the 15-HETE level increased sharply to 27.3 +/- 4.2 ng/mg protein and reached a peak of 50.0 +/- 9.8 ng/mg protein at 10 h. Ovarian 15-HETE decreased significantly between 10-16 h after hCG (when ovulation was essentially completed). The pattern of secretion of this 15-lipoxygenase product was reciprocal to the pattern of secretion of leukotriene-B4 by the rat ovary. Ovarian 15-HETE production and ovulation were inhibited in a dose-dependent manner when indomethacin was administered sc 1 h after hCG in doses ranging from 0.10-10.0 mg/rat. In contrast, the synthesis of ovarian prostaglandins-E and -F was inhibited by a dose of indomethacin as low as 0.0316 mg/rat, but this dose did not significantly affect the ovarian 15-HETE level or the ovulation rate. Therefore, the ovulation rate was more closely correlated with the ovarian 15-HETE level (P less than 0.001) than with the ovarian levels of either prostaglandin-E or -F (0.10 greater than P greater than 0.05). The results suggest that products of the 15-lipoxygenase pathway of arachidonic acid metabolism may be important in the biochemical events of mammalian ovulation.     

Temporal and hormonal regulation of inhibin protein and subunit mRNA expression by post-natal and immature rat ovaries

The contribution of specific follicle populations to dimeric inhibin production and inhibin subunit mRNA expression by the rat ovary has been investigated in two model systems, granulosa cells isolated from 25-day-old diethylstilboestrol (DES)-treated rats and post-natal rat ovaries, dispersed in culture or whole ovaries, using specific two-site immunoassays and 'real time' PCR. Media from FSH-stimulated granulosa cell cultures fractionated by gel filtration and RP-high performance liquid chromatography revealed two predominant peaks of alpha subunit activity which were attributed to alpha subunit and 31 k dimeric inhibin-A. The corresponding inhibin-B levels were low. FSH stimulation did not alter the ratio of inhibin-A:alpha subunit produced by granulosa cells. All three inhibin subunit mRNAs were expressed by granulosa cells, with eight-fold more alpha subunit mRNA relative to either of the beta subunits. Administration of DES to immature rats prior to the isolation of granulosa cells from the ovary led to beta(A) and beta(B) mRNA expression being down-regulated in the absence of any significant change in alpha subunit expression by the granulosa cells. Inhibin-A, -B and -alpha subunit were produced by basal and stimulated cultures of ovarian cells prepared from 4-, 8- and 12-day-old rats, indicating that primary, preantral and antral follicles contribute to total inhibin production. Consistent with these results, follicles within these ovaries expressed all three inhibin subunit mRNAs, with maximal expression observed in the ovaries of 8-day-old rats. The appearance of antral follicles in the ovary at day 12 led to a decline in the mRNA levels of each of the subunits but was most evident for the beta subunits. There was a profound influence of secondary preantral follicles on dimeric inhibin-A production, with FSH stimulation increasing inhibin-A relative to alpha subunit levels in cultures of ovarian cells prepared from 8-day-old rats. Thus, preantral follicles exposed to FSH contribute significantly to beta(A) subunit production by the ovary. In contrast, primary and preantral follicles did not produce inhibin-B in response to FSH stimulation. Transforming growth factor-beta (TGF-beta) enhanced, in a time-dependent manner, the production of the inhibin forms by ovarian cells in culture, although inhibin-B production was not responsive until day 8. The simultaneous treatment of ovarian cell cultures with FSH and TGF-beta elicited the greatest increases in production of all the inhibin forms. In summary, ovaries of 4-, 8- and 12-day-old rats expressed inhibin subunit mRNAs and produced dimeric inhibin-A and -B and free alpha subunit. Preantral follicles (day-8 ovarian cell cultures) were particularly sensitive to stimulation by FSH and TGF-beta and had a substantial capacity for inhibin production. The production of oestrogen by follicles may be instrumental in regulating inhibin production given that beta subunit mRNA expression was down-regulated by DES. The mechanisms by which inhibin-A and inhibin-B are individually regulated are likely to be similar during the post-natal period, when folliculogenesis is being established, and diverge thereafter, when inhibin-A becomes the predominant form in the fully differentiated ovary.     

Ovarian 3 beta-hydroxysteroid dehydrogenase and sulfated glycoprotein-2 gene expression are differentially regulated by the induction of ovulation, pseudopregnancy, and luteolysis in the immature rat.

The present studies were conducted to elucidate the effects of gonadotropin-induced alterations in ovarian status on expression of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD), an enzyme which plays a crucial role in steroidogenesis, and sulfated glycoprotein-2 (SGP-2), a heterodimeric protein which is highly expressed by cells undergoing programmed death (i.e. apoptosis). Prepubescent female rats were used to reduce the influence of endogenous gonadotropins and to avoid the presence of preexisting, degenerating corpora lutea in the ovaries. 3 beta-HSD, cholesterol side-chain cleavage cytochrome P450, and SGP-2 messenger RNA (mRNA) levels were measured by Northern analysis of total ovarian RNA. Rats which received PMSG (20 IU) followed 48 h later by human CG (hCG) (10 IU) to induce ovulation and pseudopregnancy exhibited a significant increase in ovarian 3 beta-HSD mRNA 1 day later (164%, P less than 0.01 vs. saline control). The most dramatic change in 3 beta-HSD expression was the rise seen 2 days after hCG (262%, P less than 0.01), after which levels remain constantly elevated throughout pseudopregnancy. In contrast, ovarian cholesterol side-chain cleavage cytochrome P450 mRNA was greatly elevated (i.e. 15-fold) 48 h after PMSG treatment alone (P less than 0.01). Thus, gonadotropic stimulation which induces ovulation and luteogenesis is required to observe a potent stimulatory effect on ovarian 3 beta-HSD expression. The slow time course of induction is indicative of a differentiation-dependent expression. These observations are consistent with luteal cell expression of the 3 beta-HSD gene and suggest that this expression is correlated with the high progestin secretion and 3 beta-HSD activity characteristic of luteal cells. Interestingly, the pattern of regulation of ovarian SGP-2 expression was markedly different than that observed for 3 beta-HSD. PMSG treatment alone (48 h), and in combination with hCG, dramatically reduced SGP-2 mRNA to 12-27% of controls (P less than 0.01). SGP-2 levels were not elevated until 7 days after hCG; levels then remained constant through day 14 of pseudopregnancy. Since luteal progesterone secretion begins to diminish 5-7 days after hCG, the increased expression of SGP-2 on day 7 may be related to the initiation of the regression/degeneration of luteal cells which occurs during luteolysis. Thus, this study demonstrates that alterations in SGP-2 expression by the ovary may precede or occur simultaneously with cellular events initiating luteolysis and suggests a role for this glycoprotein as an early marker for luteolysis and implicates it in yet another instance of programmed cell death.     

Expression of messenger ribonucleic acid for the antiapoptosis gene P11 in the rat ovary: gonadotropin stimulation in granulosa cells of preovulatory follicles

P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide type I receptor messenger ribonucleic acid during ovarian follicle development in the rat

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of PACAPR by gonadotropins in granulosa cells of preovulatory follicles. Moreover, RNase protection assay revealed that the short variant of ovarian PACAPR was the predominant form stimulated during prepubertal development and by gonadotropins. These results demonstrate the expression of PACAPR messenger RNA in granulosa cells of growing follicles and of preovulatory follicles stimulated by gonadotropins, and suggest that PACAP may play a role in the growth of developing follicles and in ovulation as an autocrine/paracrine factor.     

Recombinant follicle-stimulating hormone induces ovulation and tissue plasminogen activator expression in hypophysectomized rats

Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.     

Hyperstimulation and a gonadotropin-releasing hormone agonist modulate ovarian vascular permeability by altering expression of the tight junction protein claudin-5

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 microg/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 mm(2)) 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.