Preovulatory changes of blood flow in different regions of the human follicle.

OBJECTIVE: To assess regional changes in ultrasound-derived indices of blood flow in the dominant human follicle after the plasma LH surge. DESIGN: A cross-sectional, prospective study. SETTING: Reproductive medicine unit at a university. PATIENT(S): Women attending an assisted conception clinic to determine the appropriate time to transfer previously frozen embryos during a natural cycle. INTERVENTION(S): Transvaginal ultrasonography with color Doppler imaging and pulsed Doppler spectral analysis was used to obtain indices of blood flow and velocity from vessels in the base, lateral part, and apex of the dominant follicle on days 10-12 (from day 1 of menses) and after the LH surge, but before rupture. Immunoassays were used to measure the blood concentrations of LH twice daily (at 8-10 A.M. and 4-6 P.M.) from cycle day 10. MAIN OUTCOME MEASURE(S): The pulsatility index (PI), resistance index (RI), peak systolic velocity (PSV), and time-averaged maximum velocity (TAMXV) in the uterine arteries and three regions of the dominant follicle (apical, lateral, and basal parts); follicular volume; the day and time of the onset of the LH surge (defined as first concentration of LH > 22 U/L) and the times of each scan. RESULT(S): Twenty-two women (aged 28-39 years) were studied and seven were scanned on days 10-12. A retrospective examination of the data from the remainder showed that eight were scanned < 20 hours after onset of the LH surge and seven were scanned > 20 hours after the onset of the LH surge. There was a significant increase in follicular volume after the LH surge. The PI was similar in vessels from the base (0.86 +/- 0.11; mean +/- SEM), lateral part (0.72 +/- 0.51) and apex (0.67 +/- 0.09) at cycle days 10-12 and then gradually decreased in the apex. There were similar changes in the RI. The PSV (mean +/- SEM; cm/s) was similar in vessels from the base (10.1 +/- 1.64), lateral side (8.2 +/- 1.43), and apex (9.2 +/- 1.91) in follicles of days 10-12. Within 20 hours of the onset of the LH surge, the PSV had increased in basal vessels (23.4 +/- 4.10), remained similar in lateral vessels (11.64 +/- 3.18), and was undetectable in apex vessels from six of eight follicles. Twenty hours after the LH surge, there was no pulsatile blood flow observed in the apical part of the follicle, but there was a sustained high PSV in the base (15.73 +/- 3.42) and lateral side (9.02 +/- 1.5). There were corresponding changes in the TAMXV. CONCLUSION(S): During the ovulatory process there are prominent changes in the regional blood flow of the follicle with a marked increase of the flow to the base of the follicle and a concomitant decrease of blood flow to the apex. These changes may be essential for the release of a mature oocyte.

Ultrasound studies of vascular and morphological changes in the human corpus luteum during the menstrual cycle.

OBJECTIVE: To determine changes in corpus luteum (CL) volume, echogenicity, vascularity, and P production relative to a positive test result for urinary LH and day 1 of next menses. SUBJECTS: Thirteen healthy volunteers (age 23 to 32 years). INTERVENTIONS: All women underwent transvaginal ultrasonography on cycle day 11 and a urinary LH self-test was used daily. The plan was to rescan all women immediately after a positive test result and then at least every 48 hours (until day 6 of the next cycle); samples of peripheral blood were taken for analysis. MAIN OUTCOME MEASURES: The times of follicular rupture, a positive urinary LH test, and the start of menses; CL volume and echogenicity, maximum peak systolic velocity and minimum impedance, the circulating levels of serum P, E2, LH, and FSH. RESULTS: Nine women fulfilled criteria for an ovulatory cycle. There was a good correlation between peak systolic velocity, CL volume, and the concentration of serum P from day 4 to 10 after a positive LH test. Peak systolic velocity reached a maximum value between days 7 and 9 relative to a positive urinary LH test and started to decline from day 1 of menses minus 3, 4 days. CONCLUSION: Changes in peak systolic velocity from the time of a positive urinary LH self-test might be a useful adjunct for monitoring CL function.

Bradykinin potentiates LH-induced follicular rupture in the rat ovary perfused in vitro.

The possible role of bradykinin as a modulator of LH-induced ovulation was investigated using a model of the in-vitro perfused rat ovary. Ovaries from immature rats, primed with pregnant mare's serum gonadotrophin (PMSG; 20 IU), were perfused in vitro for 20 h, starting on the morning of induced proestrus. Stimulation in vitro with luteinizing hormone (LH; 0.1 microgram/ml) resulted in 3.4 +/- 1.2 ovulations per treated ovary, whereas no ovulations occurred in the unstimulated group. Bradykinin (5 microM) added to the perfusion system at 0, 2.5, 5, 7.5 and 10 h gave two ovulations in one ovulating ovary out of five ovaries perfused. When LH was combined with bradykinin, added to concentrations of 1 microM and 5 microM at the above-mentioned five time points, the numbers of ovulations were 12.2 +/- 2.7 and 15.6 +/- 3.7 per treated ovary, respectively. Bradykinin (5 microM), administered as a single dose concomitantly with LH, resulted in no further increase in the ovulation rate (3.6 +/- 1.6). Bradykinin did not affect cyclic AMP or steroid release from unstimulated or LH-stimulated ovaries. These data indicate a role of bradykinin in the ovulatory process of the rat, potentiating LH-induced ovulations.

Bradykinin induces contractions of the human ovarian follicular wall in vitro.

Bradykinin has been shown to have a positive modulatory role in the ovulatory process. In the present study, the contractile properties elicited by bradykinin (10(-12)-10(-5) M) in the isolated human follicular wall were examined. Strips from the follicular base and apex were mounted separately and superfused at 37 degrees C with oxygenated HEPES buffer in tissue chambers. The contractile activity was recorded isometrically by a force transducer (Grass model FT03), under a passive tension of 5 mN. Preovulatory follicles (cycle day 10-14) were more sensitive to bradykinin than follicles of an earlier developmental stage (cycle day 1-9). The response to bradykinin was not altered by the addition of indomethacin (10(-7) M), atropine (10(-6) M) or phenoxybenzamine (10(-7) M). These data suggest that the contraction caused by bradykinin is a specific effect, which, under physiological conditions, might contribute to the ovulatory process by inducing a rise in tone in the follicle thereby facilitating the extrusion of the oocyte through the digested follicular apex.

A prostacyclin analogue, iloprost, augments the ovulatory response of the LH-stimulated in vitro perfused rat ovary.

Ovaries from immature rats, primed with pregnant mare's serum gonadotropin (PMSG; 20 IU, on day 28), were perfused in vitro in a recirculating system for 21 h from the morning of day 30 of age. Stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) in vitro at 0 h of perfusion resulted in 2.4 +/- 0.75 (mean +/- SEM) ovulations per treated ovary, whereas no ovulations occurred in the unstimulated group. When the addition of LH was supplemented hourly for 10 h with a stable prostacyclin analogue, Iloprost, at concentrations of 0.01 microM or 0.1 microM, the ovulation rate increased significantly (p less than 0.05) to 6.6 +/- 1.3 and 10.2 +/- 2.4 ovulations per treated ovary, respectively. Iloprost (0.1 microM) did not cause any follicular ruptures when added by itself at every hour up to 10 h. The addition of Iloprost did not affect the release of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone or estradiol from unstimulated or LH-stimulated ovaries. All ovulated oocytes had resumed meiosis as judged from the absence of a germinal vesicle. These data indicate a positive modulatory role of prostacyclin in the LH-induced ovulatory process for the rat.

Neutrophil-mediated post-ischemic tubular leakage in the rat kidney.

Neutropenia was induced in male Sprague-Dawley rats by administration of antineutrophil serum (ANS). A control group received an equal volume of inactive serum. After 45 minutes of unilateral complete renal ischemia the renal blood flow (RBF) was measured by an electromagnetic flow meter. The net filtration force (NFF) in glomerular capillaries, single nephron filtration rate (SNGFR) and frequency of tubular obstructions were estimated by a micropuncture technique. Tubular leakage was measured from the fractional recovery in the normal contralateral kidney of 3H- or 14C-inulin injected into surface proximal and distal tubules of the post-ischemic kidney. Neither ANS nor inactive serum had any influence on inulin clearance (CIn) in the normal kidney. In the post-ischemic kidney, CIn was four times higher in ANS-treated than in control animals. There was no difference in RBF, NFF, SNGFR or the frequency of tubular obstructions between neutrophil-depleted and control animals. The transtubular leakage of inulin injected into proximal tubules was substantially less in the ANS-treated than in the control group (11.3 +/- 1.5% vs. 35.1 +/- 6.5%; P less than 0.01). But distal tubular leakage was equal in the two groups. The control group showed isothenuria (350 +/- 29 mOsm.kg-1), while ANS-treated animals produced hyperosmolar urine (555 +/- 60 mOsm.kg-1; P less than 0.05). It is concluded that neutrophil granulocytes mediate post-ischemic tubular leakage, which contributes to the depression in renal clearance parameters and the inability to produce hyperosmolar urine.

Evidence that the establishment of pregnancy requires activation of lipoxygenase and phospholipase-A2.

The present work investigates the possibility that lipoxygenase products are involved in the biochemical mechanisms of blastocyst implantation by utilizing nordihydroguaiaretic acid (NDGA) and caffeic acid (CA), inhibitors of lipoxygenase enzymes, and quinacrine (QU), an inhibitor of phospholipase-A2. It has been shown previously that inhibition of cyclooxygenase results in blockade of implantation. The inhibitors were dissolved in a standard medium and 5 microliter of the solutions were micro-injected into the uterine horns of day-4 pregnant mice. The contralateral horns acted as controls and received only vehicle. A sham-operated group provided normal controls. In 14 NDGA-treated mice, the control horns contained 40 implantations while the treated horns contained only 6 small implantations and 8 resorbing sites. These control horns were comparable to the sham controls. In 14 CA-treated mice, treated horns contained 17 small implantations plus 4 resorptions, whereas the control horns contained 26 small implantations and 4 resorptions. Twelve QU-treated mice exhibited 7 small implantations and 4 resorptions in the treated horns, plus 24 small sites and no resorptions in the control horns. Fourteen sham-operated mice had 95 implantation sites and no resorptions in their 28 horns. The results provide evidence for the involvement of the lipoxygenase enzymes and phospholipase-A2 in the initial implantation process and in the subsequent development of early pregnancy.

Nephron function in the early phase of ischemic renal failure. Significance of erythrocyte trapping.

Trapping of red blood cells (RBCs) in renal medulla vasculature in postischemic acute renal failure (ARF) was found to depend upon the length of the ischemic period. Thus trapping occurred after 45 minutes but not 25 minutes of ischemia. By prior hemodilution to a hematocrit (hct) of 30%, RBC trapping after 45 minutes of ischemia could be completely prevented. Likewise hemo-concentration (hct = 60%) before 25 minutes of ischemia resulted in extensive RBC trapping. By increasing or decreasing the hct, the contribution of RBC trapping to the functional defects and decrease in renal blood flow that follows minor (25 min) and more substantial (45 min) ischemia was investigated. Renal blood flow (RBF) was measured by microspheres, and vascular and tubular pressure by the micropuncture technique. Glomerular filtration rate (GFR) was estimated from inulin clearance, and tubular function from urine osmolality and sodium and potassium excretion. It was found that postischemic RBF was not correlated to RBC trapping but depended on the length of ischemia. After both 25 and 45 minutes of ischemia tubular obstructions occurred in the proximal tubules and/or loops of Henle, causing an increase in proximal tubular pressure. These obstructions were dependent on the length of ischemia but not on RBC trapping. After hemoconcentration and 25 minutes of ischemia there was an increment in distal tubular pressure, indicating that abundant RBC trapping may contribute to an increase in tubular pressure by compression of medullary tubules and thereby reduce GFR. When the damage was more severe other factors came into play and the contribution of RBC trapping to the decrease in GFR was minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

Red cell trapping after ischemia and long-term kidney damage. Influence of hematocrit.

The influence of the hematocrit (Hct) on the trapping of red blood cells (RBC) in the renal microvasculature and its effect on the long-term outcome following unilateral ischemia were investigated in the rat. The results showed that an increase in the duration of ischemia increased the RBC trapping, as measured by 51Cr-labeled erythrocytes, in a dose-dependent manner. At normal Hct (46%) the period of ischemia producing half-maximum RBC trapping was 45 minutes, whereas after hemodilution (Hct = 31%) or hemoconcentration (Hct = 60%) the corresponding periods were 80 and 25 minutes, respectively. Regarding the long-term outcome, 45 minutes of ischemia with a normal Hct was associated with a marked decrease in kidney weight, GFR and urine osmolarity after four weeks of recovery, which could be prevented to a large extent by hemodilution. Conversely, with hemoconcentration there was severe damage after only 25 minutes of ischemia. It is suggested that these long-term effects are attributable to RBC trapping in the microvasculature of the outer medulla, which may cause added ischemia in this area of the kidney. It is also suggested that cortical atrophy is secondary to the medullary injury, and is brought about to avoid extensive water and salt losses.

Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary.

Ovaries from eCG-primed (20 IU s.c. on Day 28) rats were perfused from the morning of Day 30 of age in a recirculating system initially containing a buffered blood cell-free medium (M199 + 4% BSA) for periods of up to 21 h. The addition of ovine LH (0.1 micrograms/ml) at 0 h of perfusion resulted in ovulations in all 6 ovaries perfused (3.2 +/- 0.7 ovulations per treated ovary; mean +/- SEM), whereas none of the 6 control ovaries ovulated. Rat leukocytes (50 x 10(6)), added at 7 h of perfusion significantly increased the number of LH-induced ovulations (7.8 +/- 0.5; p less than 0.05). All ovulated oocytes showed resumption of meiosis as judged from the presence of germinal vesicle breakdown. Ovaries perfused with leukocytes but without LH did not ovulate. Histological examination of ovaries 14 h after leukocyte administration showed a considerable number of perifollicular extravasated white blood cells. These findings indicate that leukocytes participate in the normal ovulatory process as part of an inflammation-like reaction.

Comparative study of transvaginal sonography and hysteroscopy for the detection of pathologic endometrial lesions in women with postmenopausal bleeding.

Dilatation and curettage is used as the "gold standard" for diagnosing pathologic endometrial lesions in women with postmenopausal bleeding. In this group of women, about 10% have an endometrial cancer and an additional 20% have some other endometrial abnormality. However, some abnormalities, such as endometrial polyps and submucous fibroids, are difficult to diagnose by dilatation and curettage. In such cases, combining transvaginal sonography with hysteroscopy may be of value. This study compared the use of transvaginal sonography and hysteroscopy for evaluation of the uterine cavity in women with postmenopausal bleeding. The study included 51 women, 39 of whom had an abnormally thick ( > 4 mm) endometrium as measured by transvaginal sonography, and 35 of 39 had an abnormal appearance at hysteroscopy. The sensitivity and specificity for the measurement of endometrial thickness using transvaginal sonography to diagnose an endometrial abnormality were 100% and 75%, respectively. The corresponding figures for hysteroscopy were 97% and 88%. In all women with an endometrial thickness of 8 mm as measured by transvaginal sonography, hysteroscopy is identified as an abnormality. The study indicates that transvaginal sonography reveals an endometrial thickness of > or = 8 mm and the histopathologic diagnosis after dilatation and curettage is atrophic endometrial polyp or submucous myoma.

Regulation of the inducible form of prostaglandin endoperoxide synthase in the perfused rat ovary.

The regulation of the two isoforms of prostaglandin endoperoxide synthase (PGS-1 and PGS-2) and prostaglandin synthesis by luteinizing hormone (LH)/3-isobutyl-1-methylxanthine (IBMX) and progesterone was examined in granulosa cells and residual ovarian tissue of rat ovaries perfused in vitro. The endogenous progesterone synthesis was blocked by an inhibitor of 3 beta-dehydroxysteroid-dehydrogenase, compound A (CA), previously shown to reversibly inhibit ovulation in the in vitro perfused rat ovary. Preovulatory ovaries were perfused for 7 h, and soluble extracts from granulosa cells and residual ovarian tissue were obtained for immunoblotting and determination of the tissue contents of PGS-1/PGS-2. The tissue concentrations of prostaglandins (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) were measured. The ovaries were perfused with medium alone (control) or medium containing LH (0.1 microgram/ml) and IBMX (0.2 mM), LH+IBMX+CA (10 micrograms/ml) or LH+IBMX+CA+progesterone (10 micrograms/ml). PGE2, PGF2 alpha and 6-keto-PGF1 alpha tissue concentrations were increased by LH+IBMX, with highest values detected for PGE2. The addition of CA alone or CA in combination with exogenous progesterone, did not change the values of prostaglandins increased by LH+IBMX. The content of PGS-1 was only marginally changed in both granulosa cells and residual ovarian tissue in the different treatment groups, compared to the control group. In contrast, PGS-2 was markedly increased by LH+IBMX, especially in the granulosa cells. The addition of CA, in combination with LH+IBMX, resulted in a small decrease of PGS-2, and progesterone further decreased its content. In the residual ovarian tissue, only minor changes of PGS-2 were detected. These results demonstrate that LH and progesterone selectively regulate the expression of PGS-2 in rat granulosa cells, whereas the hormonal regulation of PGS-1 is less pronounced. Progesterone inhibits PGS-2 in granulosa cells but has negligible effects on the total ovarian synthesis of prostaglandins during the ovulatory period.

Peritubular capillary permeability and intravascular RBC aggregation after ischemia: effects of neutrophils.

The influence of neutrophils on peritubular capillary permeability and intravascular red blood cell (RBC) aggregation after renal ischemia was studied in anesthetized Sprague-Dawley rats. Intraperitoneal administration of antineutrophil serum (ANS) reduced the number of neutrophils in the blood to 3% of normal. The control group received an equal volume of inactive serum. Renal macromolecular capillary permeability was studied from 1) extravasation of albumin and 2) plasma to lymph transport of plasma proteins and of neutral and negatively charged lactate dehydrogenase (LDH). The net driving force (NDF) for fluid transfer over the peritubular capillary membrane was determined by the micropuncture technique. The intrarenal distributions of neutrophils and RBC were measured by a histochemical method and 51Cr-labeled RBC, respectively. Under preischemic control conditions neither macromolecular permeability nor renal clearance of inulin was affected by ANS. However, the steep increase in the macromolecular transport from plasma to lymph resulting from 45 min of ischemia and reperfusion was blunted by ANS, and preischemic control values were restored after 1 h of recirculation. In the control group the mass transport of plasma proteins increased twofold and that of both neutral and negatively charged LDH fourfold. NDF was equal in the two groups. In the ANS-treated animals the intrarenal neutrophil content was only 2% of the control. Neutrophils were found mainly in the cortex, whereas RBC aggregation was observed only in the renal medulla. It is concluded that neutrophils mediate postischemic capillary leakage. It is suggested that this leakage underlies RBC aggregation and incomplete return of blood flow in the renal medulla after ischemia.

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.

Stimulatory effects of bradykinin on the ovulatory process in the in vitro-perfused rat ovary.

The role of bradykinin in the ovulatory process was investigated using an in vitro-perfused rat ovary model. Stimulation with LH (0.1 micrograms/ml) resulted in 2.6 +/- 0.5 (mean +/- SEM) ovulations per ovary, whereas no ovulations occurred in the nonstimulated control group. Bradykinin (5 microM) added to the perfusion system hourly for 10 h induced 2 of 5 ovaries to ovulate, with 2 and 3 ovulations, respectively. When bradykinin (5 microM) was given as a single dose at 5 or 10 h after LH, the ovulation rate was significantly increased to 11.0 +/- 2.8 and 8.6 +/- 2.0 ovulations per ovary, respectively. A competitive bradykinin antagonist, phenylalanine bradykinin, inhibited the bradykinin-induced increase in LH-stimulated ovulations. The addition of LH, but not of bradykinin, increased the levels of prostaglandin endoperoxide synthase in granulosa cells, but the levels of the enzyme in the residual ovarian tissue were negligible. In contrast, prostacyclin synthase was predominantly located in the residual ovarian tissue. This enzyme was not affected by LH or bradykinin. LH increased the tissue levels of prostaglandins, predominantly prostaglandin E2 (PGE2), at 7 h, whereas the stimulatory effect of bradykinin was smaller, with a preferential increase in prostacyclin (prostaglandin I2) levels. This study indicates a modulatory role of bradykinin, possibly involving prostacyclin late in the ovulatory process, in the rat.

Diverse effects of FSH and LH on proliferation of human ovarian surface epithelial cells.

The purpose of this study was to evaluate the effects of FSH and LH on growth regulation of normal ovarian surface epithelial (OSE) cells harvested from both premenopausal and postmenopausal women. Ovarian surface epithelial cells were obtained through brushing of the ovarian surface during surgery. FSH and LH were added to the OSE cultures and the proliferative effects were analysed using two different culture models, non-confluent and confluent cells, and two different detection methods, [(3)H]thymidine incorporation and a colorimetric cell number assay. FSH lowered the OSE proliferation under non-confluent conditions (10-27%), and the inhibitory effect was most pronounced among cells from postmenopausal women (P: < 0.01). In the confluent model only cells from postmenopausal women showed significantly (P: < 0.05) decreased proliferation. No effects of LH on OSE cells were detected. The unexpected results of an anti-proliferative effect of FSH on OSE, and the absence of effect by LH, do not support the theory that gonadotrophins are directly involved in ovarian carcinogenesis through an enhanced proliferation of OSE cells.

Inhibitors of lipoxygenase increase the ovulation rate in the in-vitro perfused luteinizing hormone-stimulated rabbit ovary.

In order to determine whether leukotrienes, products of the lipoxygenase pathway, are involved in ovulation, pairs of rabbit ovaries were treated with the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and caffeic acid (CA) while being perfused in vitro. The control ovaries from each rabbit received luteinizing hormone (LH) (1.5-2.25 micrograms ml-1) while the contralateral ovaries were treated with LH + NDGA (100 microM) or LH + CA (100 microM). The numbers of ovulations from both the LH + NDGA- and LH + CA-treated ovaries were significantly higher (P less than 0.05) than from their respective LH-stimulated controls. Treatment with NDGA alone in the perfusate did not cause any ovulation, while CA alone caused one ovulation from one of six ovaries perfused. Ovarian tissue levels of prostaglandins after 7 h of perfusion with LH + NDGA or with LH alone showed that, in five of the six ovaries perfused in this group, the tissue levels of PGE2, 6-keto-PGF1 alpha and PGF2 alpha were higher in the presence of NDGA. The mean differences were significant (P less than 0.05) for prostacyclin but not significant (P greater than 0.05) for PGE2 and PGF2 alpha. Our interpretation of the findings is that, when used for blocking the lipoxygenase pathway, NDGA and CA increase the substrate availability for the cyclo-oxygenase pathway of arachidonic acid metabolism, resulting in a net increase in prostaglandins. The increased ovarian levels of prostaglandins, especially prostacyclin, may cause the observed increase in ovulation rate. Consequently, although the leukotrienes may be involved in the mechanism of ovulation in the rabbit, their effects appear to be less pronounced than those of prostaglandins.

The lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibits ovulation and reduces leukotriene and prostaglandin levels in the rat ovary.

Eicosanoids, the active metabolites of arachidonic acid, are grouped into cyclooxygenase products (prostaglandins [PGs] and thromboxanes) and lipoxygenase products (leukotrienes [LTs] and lipoxins). Numerous studies suggest a role for the lipoxygenase system in ovulation. The aim of this study was to further characterize the effects of lipoxygenase inhibition and the interactions of the lipoxygenase and cyclooxygenase systems in the rat ovary during ovulation. The lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), was administered in vivo and in the isolated perfused rat ovary to determine its effect on ovulation rate. The in vivo study confirmed the inhibitory effect of NDGA, and in the perfusion experiments, NDGA caused a dose-dependent reduction in the ovulation rate. To further define the interaction between the lipoxygenase and cyclooxygenase systems, a second set of perfusions was performed with NDGA (10 microM) and the combination of NDGA (10 microM) plus a nonselective cyclooxygenase inhibitor, indomethacin (10 microM). NDGA significantly reduced the number of ovulations compared to that in controls. The ovulation rate for the combination of NDGA+indomethacin was also significantly lower than in controls but not different from that in the NDGA-treated group. Steroidogenesis was decreased only in the NDGA+indomethacin perfusions. Ovarian tissue PGE2 and PGF2alpha levels in the NDGA-treated ovaries were significantly suppressed compared to those in controls. Almost a complete block of PGE2 and PGF2alpha was seen in the NDGA+indomethacin group. LTB4 levels in the 10-h-perfused ovarian tissues were significantly decreased by NDGA compared to those in control tissues. Furthermore, LTB4 (3 microg added twice) completely reversed the inhibitory effect of 0.1 microM NDGA on ovulation rate and partially reversed the effect of 10 microM NDGA in the perfusion model. These results demonstrate that the products of the lipoxygenase pathway, especially LTB4, are important in the process of ovulation in this cyclically ovulating species. The interconnected lipoxygenase and cyclooxygenase pathways may optimize ovulation and facilitate steroidogenesis.

Saralasin-induced inhibition of ovulation in the in vitro perfused rat ovary is not replicated by the angiotensin II type-2 receptor antagonist PD123319.

OBJECTIVE: Our aim was to explain the effect of the nonspecific angiotensin II antagonist saralasin and the specific angiotensin II type-2 receptor antagonist PD123319 on ovulation. STUDY DESIGN: Saralasin, 1 micromol/L (n = 5), and PD123319 10 micromol/L (n = 6), were administered to in vitro perfused rat ovary. Prostaglandin (prostaglandin E2, prostaglandin F2alpha, 6-keto-prostaglandin F1alpha), hydroxy-eicosatetraenoic acid (12-hydroxy-eicosatetraenoic acid, 15-hydroxy-eicosatetraenoic acid), estradiol, and progesterone levels in the perfusate and the ovulation rate were compared (Mann-Whitney U test) with controls. RESULTS: Saralasin significantly (P < .01) inhibited the ovulation rate (3.0 +/- 1.4) versus control (13.1 +/- 1.0) and reduced prostaglandin E2 (at 3 hours P < .01 and 20 hours P < .05) and 6-keto-prostaglandin F1alpha (at 20 hours P < .05) levels. Saralasin did not alter prostaglandin F2alpha, hydroxy-eicosatetraenoic acids, or steroid levels. PD123319 decreased 15-hydroxy-eicosatetraenoic acid levels at 3 hours (P < .05) but had no effects on other eicosanoids, steroid levels, or the ovulation rate. CONCLUSION: Angiotensin II plays an important role in ovulation in the rat and is associated with ovarian prostaglandin synthesis. This effect is not selectively regulated via the angiotensin II type-2 receptor.

Wnt-signalling pathway in ovarian epithelial tumours: increased expression of beta-catenin and GSK3beta.

Beta-catenin is involved in both cell-cell adhesion and in transcriptional regulation by the Wingless/Wnt signalling pathway. Alterations of components of this pathway have been suggested to play a central role in tumorigenesis. The present study investigated, by immunohistochemistry and immunoblotting, the protein expression and localisation of beta-catenin, adenomatous polyposis coli (APC), glycogen synthase kinase 3beta (GSK3beta) and lymphocyte enhancer factor-1 (Lef-1) in normal human ovaries and in epithelial ovarian tumours in vivo and in vitro. Immortalised human ovarian surface epithelium and ovarian cancer cell cells (OVCAR-3) expressed beta-catenin, APC, GSK3beta and Lef-1. Nuclear staining of beta-catenin and Lef-1 were demonstrated only in OVCAR-3 cells. There were significant increases of beta-catenin and GSK3beta, while APC was reduced in ovarian cancer compared to the normal ovary. Beta-catenin and Lef-1 were coimmunoprecipitated in ovarian tumours, but not in the normal ovary. Nuclear localisation of beta-catenin or Lef-1 could not be demonstrated in the normal ovary or in the ovarian tumours. The absence of nuclear localisation of beta-catenin could be due to an increased binding to the cadherin-alpha-catenin cell adhesion complex. In fact, we have earlier reported an increased expression of E-cadherin in ovarian adenocarcinomas. In summary, this study demonstrates an increase in the expression of components of the Wingless/Wnt pathway in malignant ovarian tumours. The increase suggests a role for this signalling pathway in cell transformation and in tumour progression. However, it remains to be demonstrated whether it is an increased participation of beta-catenin in transcriptional regulation, or in the stabilisation of cellular integrity, or both, that is the crucial event in ovarian tumorigenesis.