Motomu Ando

Glucocorticoids suppress basal (but not interleukin-1-supported) ovarian phospholipase A2 activity:: Evidence for glucocorticoid receptor-mediated regulation

Ovulation may constitute a cyclic, inflammatory-like process, wherein the increased expression of interleukin (IL)-1 and the biosynthesis of prostaglandins may be established corollaries. In this communication we hypothesize that glucocorticoids, potent anti-inflammatory principles, may exert an antiovulatory effect by interfering with ovarian IL-1-driven prostaglandin biosynthesis. To test this hypothesis, we examined the effect of treatment with dexamethasone on the activity of ovarian phospholipase A2 (PLA2), the event-limiting enzyme in prostaglandin biosynthesis, and on the gene expression pattern of secretory and cytosolic PLA2 (sPLA2 and cPLA2, respectively). Whole ovarian dispersates from immature rats were cultured under serum-free conditions for 48 h in the absence or presence of dexamethasone. At the conclusion of this culture period, PLA2 activity was determined in cell sonicates and conditioned media. Parallel probing for sPLA2 and cPLA2 transcripts was also undertaken using a solution hybridization/RNAse protection assay. Treatment of whole ovarian dispersates with dexamethasone produced a significant (P < 0.005) decrease in basal cellular and extracellular PLA2 activity to 27 and 40% of controls, respectively. A 5-fold decrease in the basal steady state levels of sPLA2 (but not cPLA2) transcripts was also noted. Co-treatment with dexamethasone produced complete inhibition of IL-1-stimulated cPLA2 transcripts but not of IL-1-supported cellular and extracellular PLA2 activity or sPLA2 transcripts. A glucocorticoid receptor antagonist (RU486), blocked the ability of dexamethasone to inhibit basal sPLA2 transcripts and extracellular PLA2 activity. The inhibitory effect of dexamethasone proved glucocorticoid-specific in that aldosterone and 17beta-estradiol were without effect. Taken together, these observations suggest that dexamethasone is capable of inhibiting basal (but not IL-1-supported) ovarian PLA2 activity, a glucocorticoid receptor-mediated effect due, in part, to a decrease in sPLA2 gene expression. Our findings further suggest that sPLA2 and cPLA2 are differentially regulated and that they may well differ in their relative contribution to ovarian prostaglandin biosynthesis in general and to PLA2 activity in particular. To the extent that IL-1 plays a central role in the ovulatory process, these findings argue against the view that the chronic anovulatory state induced by glucocorticoid excess is due, if only in part, to suppression of ovarian IL-1-dependent PLA2 activity.

Treatment of uterine leiomyomata with a luteinizing hormonereleasing hormone agonist: the possibility of nonsurgical management in selected perimenopausal women

OBJECTIVE To evaluate the efficacy of luteinizing hormone-releasing hormone agonist (LH-RH-a) in the treatment of leiomyomata. DESIGN A retrospective randomized trial. SETTING Hospital department of obstetrics and gynecology. PATIENTS Twenty-five women, ages 36 to 54 years with symptomatic uterine leiomyomata, were divided into two groups according to the responsiveness to LH-RH-a: group A patients reached menopause after LH-RH-a, whereas resumption of menstruation occurred within 12 weeks after cessation of therapy in group B. INTERVENTIONS Luteinizing hormone-releasing hormone agonist was administered intranasally three times a day with 150 micrograms insufflation of one spray in each nostril (total dose: 900 micrograms/d). MAIN OUTCOME MEASURES Efficacies of treatment were assessed in terms of uterine volume, hemoglobin concentrations, serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and bone density during and after treatment. RESULTS In both groups, hemoglobin concentrations increased significantly after 16 weeks of treatment. A significant reduction in uterine volume was observed in both groups. After completing therapy, there was no further significant change in uterine volume in group A, whereas uterine volume in group B returned to pretreatment values. Serum LH and FSH concentrations were suppressed during treatment, but those gonadotropins in group A increased significantly up to the menopausal levels after treatment. Serum E2 concentrations in both groups showed consistent suppression by the end of the first treatment cycle. After cessation of therapy, serum E2 levels on group A remained in the castrate range, whereas E2 in group B returned to pretreatment levels, concomitant with the return of normal ovulation. CONCLUSIONS Intranasal administration of LH-RH-a was successful in significantly decreasing uterine volume and increasing hemoglobin concentration in premenopausal women with leiomyomata.

The rat intraovarian interleukin (IL)-1 system: cellular localization, cyclic variation and hormonal regulation of IL-1β and of the type I and type II IL-1 receptors

An increasing body of evidence supports the possibility that intraovarian interleukin (IL)-1 plays an intermediary role in the periovulatory cascade. To gain further insight into the intraovarian IL-1 hypothesis, we studied the cellular localization cyclic variation and hormonal regulation of IL-1beta, as well as of the type I and type II IL-1 receptors (IL-1R) in immature rats. In situ hybridization localized IL-1beta and type I IL-1R transcripts to the granulosa cell compartment, the innermost layers of the theca interna and to the oocyte of the untreated immature ovary. Molecular probing of whole ovarian material in the course of a simulated estrous cycle revealed a progressive preovulatory increase in IL-1beta and type I IL-1R transcripts to an in vivo peak at the time of ovulation (3.0- and 2.5-fold increases over untreated controls; P < 0.05). Comparable efforts to localize and probe for type II IL-IR transcripts failed to elicit a detectable signal. The basal in vitro expression pattern of IL-1beta and type II IL-1R transcripts by whole ovarian dispersates revealed an early (4 h) spontaneous increase to a peak (2.1- and 5.8-fold increases over time 0: P < 0.05) followed by a gradual decline to a 48 h nadir. Treatment of whole ovarian dispersates with the IL-1 receptor antagonist (IL-1RA) or with IL-1beta failed to alter the initial (4 h) burst of IL-1beta or of type II IL-1R expression thereby suggesting IL-1-independence. Treatment with hCG proved equally ineffective. However, longer-term treatment of whole ovarian dispersates with IL-1beta produced a significant secondary increase (5.9-fold over time 0; P < 0.05) in IL-1beta (but not type II IL-1R) transcripts by 48 h. This IL-1 effect was completely blocked by co-treatment with IL-1RA thereby suggesting mediation via a specific IL-1 receptor. Qualitatively comparable but quantitatively reduced results obtained for isolated granulosa cells. The basal in vitro expression pattern of type I IL-1R transcripts by whole ovarian dispersates revealed a progressive spontaneous increase (3.1-fold increase overall) over the 48 h culture. Treatment with IL-1beta produced a significant (P < 0.05) increase (5-fold) in type I IL-1R transcripts by 48 h, an effect which was completely blocked by co-treatment with IL-1RA. Taken together, these observations: (1) localize IL-1beta and its type I receptor to granulosa cells, the innermost layers of the theca interna and to the oocyte; (2) confirm their periovulatory in vivo expression pattern; (3) document their expression by untreated cultured whole ovarian dispersates; and (4) demonstrate their in vitro responsiveness to receptor-mediated/IL-1-driven autocrine amplification. The type II IL-1R was undetectable in vivo, its in vitro expression pattern proving IL-1- and hCG-independent. The periovulatory expression pattern of IL-1beta and its receptor (type I) is compatible with the notion that the intraovarian IL-1 system may play an intermediary role in the ovulatory process.

Direct effect of gonadotropin-releasing hormone agonists on the rabbit ovarian follicle

OBJECTIVE To determine if gonadotropin-releasing hormone agonist (GnRH-a)-induced oocyte maturation and degeneration can be attributed to the direct actions on the follicle. DESIGN Mature rabbit follicle culture. INTERVENTIONS The mature follicles were cultured with human chorionic gonadotropin (hCG) (100 ng/mL), buserelin acetate (10(-9) to 10(-6) M), leuprolide acetate (10(-9) to 10(-6) M), or buserelin acetate (10(-7) M) with a GnRH antagonist (10(-8) to 10(-6) M) for 14 hours. MAIN OUTCOME MEASURES The percentage of oocytes achieving germinal vesicle breakdown, the oocyte degeneration rate, prostaglandins (PG) production by mature follicles, and the frequency of fertilization and embryonic development. RESULTS Gonadotropin-releasing hormone agonist induced the meiotic maturation of follicle-enclosed oocytes in a dose-dependent manner while concomitantly increasing oocyte degeneration. The simultaneous addition of GnRH antagonist inhibited significantly GnRH-a-induced oocyte maturation and PG production by the mature follicles. Furthermore, a GnRH antagonist reversed the oocyte degeneration rate that had been increased by GnRH-a. The rates of normal fertilization and early embryonic development were significantly reduced in the oocytes matured by GnRH-a as compared with those matured by hCG. CONCLUSIONS Gonadotropin-releasing hormone agonist acts directly on mature rabbit follicles to trigger the oocytes to undergo meiotic maturation, but oocytes matured in vitro by GnRH-a are not necessarily cytoplasmically mature.

Possible Contribution of Prolactin in the Process of Ovulation and Oocyte Maturation

The present study was undertaken to evaluate the effects of PRL in the process of ovulation and oocyte maturation. In the first experiment, using an in vitro perfused rabbit ovary model, the addition of PRL to the perfusate inhibited hCG-induced ovulation in a dose-related fashion, without any reduction in progesterone synthesis. In a subsequent experiment, PRL directly inhibited both the degeneration and decomposition of surface epithelial cells and the disruption of connective tissue at the apex of the follicle wall. Furthermore, PRL inhibited hCG-stimulated plasminogen activator (PA) activity in mature follicles in a dose-related fashion. In the final experiment, we demonstrated conditions in which rabbit oocytes matured in vitro acquire competence for early embryonic development. PRL, as well as gonadotropins and estradiol, was an important constituent in the process of oocyte maturation, promoting embryonic development. These results suggest that the preovulatory environment of PRL within the follicle may influence the process of ovulation and oocyte maturation.

Gonadotropin-releasing hormone agonists induce meiotic maturation and degeneration of oocytes in the in vitro perfused rabbit ovary.

The present study was undertaken to assess the effects of gonadotropin-releasing hormone agonists (GnRH-a, buserelin and leuprolide acetate [LA]) on ovulation, oocyte maturation and degeneration, and steroid and prostaglandin production in the perfused rabbit ovary preparation. Ovulation did not occur in any of ovaries treated with buserelin or LA (10(2) to 10(4) ng/mL) in the absence of gonadotropin. Gonadotropin-releasing hormone agonists were associated with the resumption of meiosis in follicular oocytes in a dose-related manner. Furthermore, the addition of GnRH-a to the perfusate significantly increased the percentage of follicular oocytes that showed evidence of degeneration compared with contralateral untreated or human chorionic gonadotropin-treated controls. Prostaglandin E2 and prostaglandin F2 alpha production by the perfused rabbit ovaries were stimulated significantly by GnRH-a treatment. Exposure to GnRH-a failed to increase either progesterone or estradiol production by the perfused rabbit ovaries. These data demonstrate that GnRH-a act directly in the rabbit ovary to trigger meiotic maturation in oocytes within the follicles, concomitantly increasing oocyte degeneration.

Periovulatory and Interleukin (IL)-1—Dependent Reglation of IL-6 in the Immature Rat Ovary: A Specific IL-1 Receptor-Mediated Eicosanoid-Dependent Effect

Interleukin (IL)-6, traditionally an IL-1—induced immune regulator, is nevertheless synthesized by a variety of tissues including the ovary. The purposes of this communication were to assess the ovarian expression of IL-6, examine its cyclic variation, and study its regulation by IL-1, a putative intermediary in the ovulatory process. Molecular probing revealed IL-6 transcripts to be most abundant in the thymus, liver, and ovary, which suggests that, in relative terms, untreated immature whole ovarian tissue is a significant site of IL-6 gene expression. Treatment of immature rats with pregnant mare serum gonadotropins resulted in a meausrable, statistically significant (P < .05) increase in ovarian IL-6 mRNA compared with untreated controls. Isolated and indeed transient increments in the relative abundance of IL-6 transcripts were noted 4 hours after exposure to hCG, a point in time 8 hours from projected follicular rupture, a pattern highly reminiscent of that previously recognized for IL-1β transcripts. Treatment of whole ovarian dispersates from immature rats with IL-1β for 48 hours resulted in a significant (P < .05) increase (11-fold) over control in IL-6 transcripts. The IL-1 effect proved dose dependent; the first significant increase was noted at the 1-ng/mL dose level. Evaluation of the time requirements revealed IL-1β to significantly (P < .05) up-reglate (4.5-fold) IL-6 transcripts as early as 24 hours into the culture period. Cotreatment with the IL-1 receptor antagonist completely reversed the IL-1 effect, which suggests mediation through a specific IL-1 receptor. Treatment with indomethacin (10 μg/mL), an established inhibitor of prostaglandin biosynthesis, resulted in a significant (P < .05) decrease (79%) in the ability of IL-1β to up-regulate IL-6 transcripts. Importantly, the addition of prostaglandin E2 (10 μg/mL) to untreated or indomethacin-treated cells significantly (P < .05) augmented the IL-1β effect. This suggests a role for eicosanoid signaling in IL-1β action. Treatment with aminoguanidine, an established inhibitor of nitric oxide synthase, significantly (P < .05) decreased (85%) the IL-1β effect. However, the addition of S-nitroso-n-acetyl-penicilamine, an established nitrite generator, failed to reverse the aminoguanidine effect, which suggests that the inhibitor effect of aminoguanidine may be nitrite independent. Treatment with cycloheximide produced dose-dependent inhibition of the ability of IL-1 to up-regulate IL-6 transcripts; the maximal inhibitory effect was 89%. Taken together, these findings (1) reaffirm the rat ovary as a site of IL-6 expression; (2) document an in vivo increase in IL-6 transcripts before ovulation; (3) disclose a marked dependence of IL-6 on IL-1β; and (4) reveal the IL-1β effect to be dose and time dependent, receptor mediated, contingent upon de novo protein biosynthesis, and eicosanoid dependent but nitric oxide independent. These findings suggest that IL-1 action in the ovary may require the intemediacy of IL-6 in a manner like that encountered in extraovarian sites.

Direct Ovarian Effect of Growth Hormone in the Rabbit

PROBLEM: This study was undertaken to assess whether growth hormone (GH) can stimulate follicle growth and ovarian steroidogenesis via putative GH receptors.

Possible Involvement of Lipoxygenase Products in Human Corpora lutea

The present study was undertaken to determine the ability of cultured luteal cells from human corpora lutea to secrete progesterone (P4) and prostaglandins (PGs), and to assess the effects of the products of the lipoxygenase pathway on luteal P4 production. Luteal cells responded to human chorionic gonadotropin (hCG) with a significant increase (2- to 7-fold) in P4 production. Arachidonic acid significantly stimulated PGE2 synthesis by luteal cells in a dose-dependent manner. Both basal PGE2 production and the responsiveness to arachidonic acid were maintained for 8 days. In contrast, both PGF2 alpha and 6-keto-PGF1 alpha production abruptly declined as the culture proceeded. However, the addition of hCG did not further stimulate the accumulation of the 3 PGs assayed. In the subsequent experiment, 5-hydroxyeicosatetraenoic acid (5-HETE) and the reaction products of soybean lipoxidase of arachidonic acid (AA-LIP) were utilized for evaluating the involvement of the lipoxygenase pathway in luteolysis. The addition of 5-HETE dose-dependently inhibited P4 production by the cultured luteal cells. Although treatment with either arachidonic acid or lipoxidase alone had no effect on P4 production, AA-LIP significantly reduced P4 production in the presence or absence of hCG. These results suggest that the products of the lipoxygenase as well as of the cyclo-oxygenase pathway may be important in regulating the life span and function of human corpora lutea.