Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470.

Angiogenesis, the growth of new blood vessels, occurs normally in female reproductive organs. We tested the hypothesis that angiogenesis inhibition may affect fertility by studying the reproductive system in either pregnant or nonpregnant cycling mice after treatment with the angiogenesis inhibitor AGM-1470. Administration of AGM-1470 to pregnant mice resulted in complete failure of embryonic growth due to interference with decidualization, placental and yolk sac formation, and embryonic vascular development. When nonpregnant cycling female mice were chronically treated with AGM-1470, inhibition of endometrial maturation and corpora lutea was observed. These data suggest that processes in reproduction can be controlled through angiogenesis inhibition.

Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo.

Elevated vascular endothelial growth factor (VEGF) levels are required for ocular and tumor angiogenesis in animal models. Ischemic hypoxia is strongly correlated with increased VEGF expression in these systems and is considered a physiologically relevant stimulus. Because ischemic hypoxia is often followed by reperfusion and reactive oxygen intermediate (ROI) generation, we examined the potential role of ROI in the control of VEGF gene expression. Human retinal pigment epithelial cells exposed to superoxide or hydrogen peroxide rapidly increased VEGF mRNA levels. Superoxide-associated mRNA increases were dose dependent, blocked by antioxidants, and associated with elevated VEGF protein levels in conditioned media. Increases in VEGF mRNA levels were also observed in cultured human melanoma and rat glioblastoma cells with superoxide or hydrogen peroxide. Cycloheximide prevented the ROI-associated increases in VEGF mRNA. Transcriptional inhibition with actinomycin D revealed an inducible increase in VEGF mRNA half-life, but nuclear run-on experiments showed no increase in VEGF transcriptional rate. Reoxygenation of human retinal pigment epithelial cells in vitro and ocular reperfusion in vivo increased retinal VEGF mRNA levels. Antioxidants prevented the reperfusion-associated VEGF mRNA increases in retina. We conclude that ROIs increase VEGF gene expression in vitro and during the reperfusion of ischemic retina in vivo. The ROI-associated increases are mediated largely through increases in VEGF mRNA stability.

The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process.

This study characterizes the rat ovary as a site of hormonally dependent glucose transporter (Glut) expression, and explores the potential role of interleukin (IL)-1, a putative intermediary in the ovulatory process, in this regard. Molecular probing throughout a simulated estrous cycle revealed a significant surge in ovarian Glut3 (but not Glut1) expression at the time of ovulation. Treatment of cultured whole ovarian dispersates from immature rats with IL-1beta resulted in upregulation of the relative abundance of the Glut1 (4.5-fold) and Glut3 (3.5-fold) proteins as determined by Western blot analysis. Other members of the Glut family (i.e., Gluts 2, 4, and 5) remained undetectable. The ability of IL-1 to upregulate Glut1 and Glut3 transcripts proved time-, dose-, nitric oxide-, and protein biosynthesis-dependent but glucose independent. Other ovarian agonists (i.e., TNF alpha, IGF-I, interferon-gamma, and insulin) were without effect. Taken together, our findings establish the mammalian ovary as a site of cyclically determined Glut1 and Glut3 expression, and disclose the ability of IL-1 to induce the ovarian expression as well as translation of Glut1 and Glut3 (but not of Gluts 2, 4, or 5). Our observations also establish IL-1 as the first known regulator of Glut3, the most efficient Glut known to date. In so doing, IL-1, a putative component of the ovulatory process, may be acting to meet the increased metabolic demands imposed on the growing follicle and the ovulated cumulus-enclosed oocyte.

Intraovarian regulation: peptidergic signaling systems.

Intraovarian regulation, an evolving field, is now at a crossroad. Although a number of putative intraovarian regulators appear to be of import to ovarian physiology, none has thus far been demonstrated to be indispensable to in vivo ovarian function. That notwithstanding, it is already clear that optimal gonadotropin hormonal action is highly contingent upon the input of tissue-based regulatory principles. It is with a strong sense of excitement that future work in this evolving area is anticipated.

Endocrine- and autocrine-mediated regulation of rat ovarian (theca-interstitial) interleukin-1 beta gene expression: gonadotropin-dependent preovulatory acquisition.

We, and others have recently demonstrated the ovary to be a site of interleukin-1 (IL-1) reception and action. Since IL-1 is an established mediator of inflammation and since ovulation may constitute an inflammatory-like reaction, consideration was given to the possibility that IL-1 may play an intermediary role in the ovulatory process. To begin to evaluate the above hypothesis, we have set out to evaluate rat ovarian IL-1 beta gene expression, to determine its cellular localization, and to study its modulation by key endocrine and autocrine regulatory signals. To this end, use was made of a solution hybridization/RNase protection assay in which rat ovarian total RNA (20 micrograms) was hybridized with a [32P]-labeled 272 base rat IL-1 beta antisense riboprobe. To assess rat ovarian IL-1 beta gene expression under in vivo circumstances, use was made of an established experimental model capable of simulating naturally-occurring follicular maturation, ovulation, and corpus luteum formation. Specifically, a single subcutaneous injection of PMSG (15IU/rat) was followed (48h) later by an ovulatory dose (15IU) of human chorionic gonadotropin (hCG). A faint protected fragment 222 bases long corresponding to the IL-1 beta message was detectable in whole ovarian material prior to gonadotropic stimulation. Treatment with PMSG for 48h resulted in a modest, albeit measurable increase in the densitometrically-quantified steady state levels of the ovarian IL-1 beta message. Most striking, however, were the increments noted in the relative abundance of ovarian IL-1 beta transcripts following a 6h exposure to hCG producing a 4 to 5-fold increase (P less than 0.05) over the untreated state at a time point approximately 6h prior to projected follicular rupture. Subsequent evaluation of ovarian IL-1 beta transcripts, 24 and 48h following hCG administration, revealed significant (P less than 0.05) decrements (relative to the 6h peak) to a level comparable to that seen at the conclusion of 48h of treatment with PMSG. Cellular localization studies revealed the gonadotropin-dependent IL-1 beta mRNA to be theca-interstitial cell-exclusive. To assess rat ovarian IL-1 beta gene expression under in vitro circumstances, we have set out to determine whether IL-1 itself may influence the relative level of its own message. Treatment of whole ovarian dispersates with rhIL-1 beta (10ng/ml) for 4 and 24h resulted in a marked (P less than 0.05) time-dependent increase (up to 12-fold) in the relative abundance of IL-1 beta transcripts when compared with untreated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and relative abundance of alternatively spliced luteinizing hormone receptor messenger ribonucleic acid in the ovine ovary.

Complementary DNA (cDNA) clones encoding the LH receptor (LHR) were recently isolated from pig, rat, mouse, and human testes or ovaries. Many of the LHR cDNAs isolated from these species encoded incomplete and, therefore, possibly inactive forms of the LHR. The four major incomplete cDNAs, designated B, C, D, and E, were due to alternative splicing of the full-length cDNA, designated the A form. Northern analyses of messenger RNA (mRNA) encoding LHR in these species and in sheep revealed multiple mRNA species in ovarian tissue, but were unable to distinguish between the full-length (functional) form and the splice variants. We have used reverse transcription of mRNA, amplification via the polymerase chain reaction, and cDNA sequencing to determine which alternatively spliced mRNA species were present in ovine ovarian follicles and corpora lutea, and ribonuclease protection assays to confirm these results and determine the relative abundance of these splice variants. Ovine LHR cDNAs of the full-length A form, B form, and two novel splice forms, designated F and G, were isolated and sequenced. By using LHR cDNAs that spanned the regions of the gene in which the majority of splicing variation occurred, ribonuclease-protected fragments of different sizes were generated depending on which mRNA species (A-G) were present. It is estimated that the ratios of the steady state mRNA levels of the splice variant B form/full-length A form/G form/F form were 5-3.5:1:1:0.3. The E, C, and D forms were not detected, even when using the sensitive method of reverse transcription-polymerase chain reaction for the latter two forms. The overall level of expression of LHR mRNA was greater in corpora lutea than follicles, but the relative abundance of the splice variants was similar in follicles and corpora lutea.

Rat ovarian insulin-like growth factor-binding protein-6: a hormonally regulated theca-interstitial-selective species with limited antigonadotropic activity.

Most but not all of the established insulin-like growth factor-binding proteins (IGFBPs) are expressed in the rat ovary. To continue the process of characterizing these ovarian IGFBPs, a solution hybridization/RNase protection assay was used to study IGFBP-6 gene expression, cellular localization, and hormonal regulation in the immature rat ovary. Total RNA was hybridized with a 458-base long 32P-labeled rat IGFBP-6 cRNA. A single protected fragment (380 bases long) corresponding to IGFBP-6 transcripts was identified in RNA from ovary and lung, but not kidney or liver. The amount of IGFBP-6 transcripts was higher in ovaries from immature rats (25-28 days old) than in ovaries from adult rats and was higher in theca-interstitial than in granulosa cell preparations. Hypophysectomy resulted in a significant (P < 0.05) 2.3 +/- 0.7-fold (mean +/- SD) increase in whole ovarian IGFBP-6 transcripts. This suggests that ovarian IGFBP-6 gene expression is subject to inhibition by one or more pituitary hormones. Therefore, the effect of replacement of FSH, GH, diethylstilbestrol (DES), or combinations thereof was evaluated. Treatment with FSH resulted in a 2.4-fold decrease (P < 0.05) in the abundance of ovarian IGFBP-6 transcripts relative to that in hypophysectomized controls. Provision of DES yielded comparable results. Moreover, the combined provision of FSH and DES resulted in a synergistic decrease (6.0-fold) in ovarian IGFBP-6 transcripts. In contrast, treatment of hypophysectomized rats with GH proved without effect. However, treatment with FSH or DES in addition to GH resulted in a decrease in ovarian IGFBP-6 transcripts (3.9- and 2.7-fold, respectively). To assess the role of ovarian IGFBP-6, its influence on gonadotropin action in primary cultures of rat granulosa cells was also studied. Increasing concentrations (0.01-1 micrograms/ml) of recombinantly expressed human IGFBP-6 did not inhibit the FSH-supported accumulation of either progesterone or estradiol. These findings 1) establish the theca-interstitial compartment of the immature rat ovary as a prominent site of IGFBP-6 gene expression, 2) implicate FSH and DES as inhibitors of IGFBP-6 transcript levels in the whole ovary, 3) and disclose the limited antigonadotropic action of IGBP-6F on the rat granulosa cell.

Rat ovarian prostaglandin endoperoxide synthase-1 and -2: periovulatory expression of granulosa cell-based interleukin-1-dependent enzymes.

This laboratory has previously shown that interleukin-1 (IL-1), a putative intermediary in the ovulatory process, is capable of up-regulating PG biosynthesis by cultured whole ovarian dispersates from immature rats. In part, this phenomenon was attributable to the stimulation of ovarian phospholipase A2 activity. In this communication we examine the possibility that the PG-promoting property of IL-1 is also due to the up-regulation of PG endoperoxide synthase (PGS), the rate-limiting step in prostanoid biosynthesis. The in vivo expression of ovarian PGS-2 transcripts in the course of a simulated estrous cycle rose abruptly to a peak (35-fold increase over the control value; P < 0.05) 8-12 h after hCG administration (i.e. before or during projected ovulation). PGS-1 transcripts, in turn, were not significantly altered during the periovulatory period. Treatment of cultured whole ovarian dispersates with IL-1beta resulted in dose- and time-dependent up-regulation of PGS-2 transcripts (as well as of immunoreactive PGS-2 protein and PGE2 accumulation), characterized by an ED50 of 2 ng/ml and a maximal (72-fold) increase at 10 ng/ml. Although treatment with IL-1beta also led to an increase in PGS-1 transcripts and immunoreactive PGS-1 protein, the relative magnitude of the effect was markedly reduced compared with that of PGS-2. Cotreatment with an IL-1 receptor antagonist completely reversed the IL-1 effects, thereby suggesting mediation via the IL-1 receptor. The ability of IL-1 to up-regulate PGS-2 transcripts proved relatively specific, in that other cellular regulators (insulin-like growth factor I, activin A, endothelin-1, transforming growth factor-alpha, tumor necrosis factor-alpha, vascular endothelial growth factor, leukemia inhibitor factor, hepatocyte growth factor, or keratinocyte growth factor) were not effective. The optimal IL-1 effect required heterologous contact-dependent coculturing of granulosa and thecal-interstitial cells. Taken together, these observations 1) reaffirm (by molecular probing) the granulosa cell as the primary site of ovarian PGS-1 and PGS-2 expression, 2) document an increase in ovarian PGS-2 transcripts before ovulation, and 3) reveal a marked dependence of ovarian PGS (2 >> 1) transcripts, proteins, and activity on IL-1. The effects of IL-1 proved relatively specific, contingent upon somatic cell-cell cooperation, dose and time dependent, and IL-1 receptor mediated. These results are compatible with the proposition that the PG-promoting property of IL-1 is due, in large measure, to the activation of ovarian PGS transcription and translation. The ability of IL-1 to up-regulate ovarian PGS, an obligatory component of ovulation, is in keeping with the idea that IL-1 may constitute an intermediary in the ovulatory process.

Rat ovarian insulin-like growth factor binding protein-3: a growth hormone-dependent theca-interstitial cell-derived antigonadotropin.

To further the identification and characterization of insulin-like growth factor binding proteins at the level of the immature rat ovary, we have set out to study the ovarian expression, cellular localization, and hormonal regulation of the insulin-like growth factor binding protein (IGFBP)-3. To this end, use was made of a solution hybridization/RNAse protection assay wherein ovarian total RNA from immature (21-23 days old) female rats was hybridized with a 343 bases-long [32P]-labeled rat IGFBP-3 riboprobe. As in liver, a single protected fragment (315 bases-long) corresponding to IGFBP-3 transcripts was identified in whole ovarian material. Cellular localization studies revealed the IGFBP-3 gene to be exclusively expressed in the theca-interstitial rather than the granulosa cell compartment. To confirm presence and cellular distribution of the IGFBP-3 protein, media conditioned by cultured granulosa cells, theca-interstitial cells, and whole ovarian dispersates were subjected to Western Ligand Blotting. Importantly, media conditioned by cultured theca-interstitial (but not granulosa) cells displayed an IGFBP the size of rat IGFBP-3 (46kDa) as determined by comigration with a rat serum standard. A similarly-sized band was apparent in media conditioned by cultured whole ovarian dispersates reflecting in all likelihood the contribution of the theca-interstitial cell component. Significantly, deglycosylation of media conditioned by cultured theca-interstitial cells revealed the glycosylated nature of the 46kDa IGFBP species as judged by the apparent reduction in its molecular size to 35kDa. Similar alterations were noted in corresponding rat serum samples. Hypophysectomy of immature rats resulted in a modest but statistically insignificant decrease in the relative (densitometrically-quantified) abundance of ovarian IGFBP-3 transcripts, an effect further augmented by the systemic provision of either FSH or diethylstilbestrol (DES). In contrast, systemic treatment of hypophysectomized rats with GH produced a marked (3.2-fold) increase (P less than 0.05) in the steady state levels of ovarian (as well as hepatic) IGFBP-3 transcripts. However, the concurrent provision of either FSH or DES resulted in substantial (P less than 0.05) attenuation (78 and 57% inhibition, respectively) of the upregulatory GH effect. These findings document the highly compartmentalized expression of the IGFBP-3 gene at the level of the immature rat ovary, implicate the theca-interstitial cell as the sole source of its generation, reveal its pituitary dependence, and disclose its diametrically-opposed (indeed antagonistic) regulation by FSH (or estrogens) and GH.(ABSTRACT TRUNCATED AT 400 WORDS)

The follicle-stimulating hormone receptor and luteinizing hormone receptor genes are closely linked in sheep and deer.

Restriction fragment length polymorphisms were identified in sheep and deer using ovine cDNA probes for the FSH receptor (FSHR) and the LH receptor (LHCGR). FSHR and LHCGR were closely linked in sheep with no recombinants and neither receptor was linked to the Booroola fecundity gene (FecB). Both receptors were also closely linked in deer at a map distance of 3.3 cM. Linkage between the receptor genes assigns FSHR to sheep chromosome 3. Sequence analysis showed that the mammalian LHCGRs and FSHRs are more similar to each other than to mammalian TSH receptor (TSHR). Taken together, these data suggest that TSHR and the LHCGR/FSHR arose from a common ancestral gene by a process of chromosomal duplication. Subsequent duplication of the region containing the LH/FSH receptor and functional divergence could have given rise to the two gonadotrophin receptors present in mammals today.

Suppression of hypoxia-associated vascular endothelial growth factor gene expression by nitric oxide via cGMP.

PURPOSE: To investigate the suppressive effect of nitric oxide (NO) on vascular endothelial growth factor (VEGF) gene expression and to elucidate its mechanism of action. METHODS: Immortalized human retinal epithelial (RPE) cells, H-ras-transfected murine capillary endothelial cells, and nuclear factor-kappaB (NF-kappaB) RelA knockout 3T3 fibroblasts had VEGF gene expression stimulated by hypoxia, TPA (phorbol ester 12-O-tetradecanoylphorbol-13 acetate), and ras-transfection. The dose response and time course of inhibition of VEGF gene expression by NO were characterized by northern blot analysis, ribonuclease protection assay, and enzyme-linked immunosorbent assay. The effects of NF-kappaB and cGMP in the NO-induced suppression of VEGF gene expression were quantitated. cGMP production was inhibited by LY 83583 (6-anilino-5,8-quinolinedione), a specific inhibitor of guanylate cyclase production, and cGMP accumulation was quantitated by immunoassay. RelA knockout 3T3 fibroblasts were used to assess the contribution of NF-kappaB to the downregulation of VEGF by NO. RESULTS: The NO donor sodium nitroprusside (SNP) decreased hypoxia-induced VEGF gene expression in a dose- and time-dependent manner. One hundred fifty micromolar SNP completely suppressed hypoxia-induced VEGF mRNA levels for at least 24 hours. Constitutive VEGF expression was not altered by SNP. The SNP-mediated decreases in VEGF expression were associated with increases in intracellular cGMP and were blocked by LY 83583. Sodium nitroprusside was able to decrease hypoxia-induced VEGF mRNA increases in fibroblasts deficient in the RelA subunit of NF-kappaB. Nitric oxide was also effective at suppressing increased VEGF expression secondan, to mutant ras and TPA. CONCLUSIONS: These data indicate that NO decreases hypoxia-induced VEGF via a cGMP-dependent mechanism and suggest that NO may serve as an endogenous inhibitor of both hypoxia- and non- hypoxia-enhanced VEGF expression in vivo.

Transforming growth factor-beta1 is a potent inhibitor of interleukin-1beta action in whole ovarian dispersates.

Transforming growth factor beta1 (TGFbeta1) acts as an inhibitor of the actions of interleukin-1beta (IL-1beta) in various organ systems. In order better to understand the inter|P-actions between these polypeptides in the ovary, we evaluated the effect of TGFbeta1 co-treatment on various IL-1beta-mediated actions in cultures of whole ovarian dispersates. Treatment with IL-1beta enhanced media accumulation of nitrites (4.8-fold), prostaglandin E2 (PGE2, 3. 9-fold) and lactate (2.0-fold), and enhanced glucose consumption (2. 1-fold). Treatment with TGFbeta1 alone did not significantly affect any of these parameters. However, the addition of TGFbeta1 inhibited IL-1beta-stimulated nitrite (100%), PGE2 (44%) and lactate (78%) accumulation and inhibited IL-1beta-stimulated glucose consumption (74%) in a dose-dependent manner. The addition of TGFbeta1 also suppressed the steady-state levels of IL-1beta-stimulated IL-1beta, type I IL-1 receptor and IL-1 receptor antagonist transcripts (98, 67 and 83% inhibition respectively). These data suggest that TGFbeta1 is capable of inhibiting several IL-1beta-stimulated endpoints. Since IL-1 has been identified as a possible proinflammatory mediator of ovulation and TGFbeta has been implicated as a promotor of fibrosis and healing, we speculate that IL-1 and TGFbeta might play antagonistic roles in the normal ovulatory sequence.

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.

The rat intraovarian interleukin (IL)-1 system: cellular localization, cyclic variation and hormonal regulation of IL-1beta 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.

In vivo hormonal regulation of insulin-like growth factor binding protein-5 mRNA in the immature rat ovary.

OBJECTIVE: Despite the potential importance of insulin-like growth factor binding protein-5 (IGFBP-5) to follicular development, the hormonal regulation of this antigonadotropic IGFBP has not been investigated. Therefore, it was the objective of this study to eludicate the role of gonadotropins and estrogen in the in vivo regulation of IGFBP-5 mRNA expression. METHODS: Two models of follicular development in immature rats were used. Specifically, rats were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Alternatively, terminal follicular development was induced in intact immature rats by pregnant mare serum gonadotropin (PMSG) and hCG. The IGFBP-5 mRNA in whole ovarian RNA was assayed by Northern blot hybridization. Localization of expression in PMSG and hCG-stimulated ovaries was further assessed by in situ hybridization. RESULTS: Expression of IGFBP-5 mRNA was increased in ovaries from hypophysectomized rats. Treatment with FSH and/or DES did not alter the abundance of this mRNA. Treatment with PMSG induced a transient increase in IGFBP-5 expression that was localized in a subset of alpha-inhibin-negative follicles. At later times after PMSG, IGFBP-5 expression persisted in the surface epithelium but was not detected in large preovulatory follicles. In vitro studies affirmed the antigonadotropic action of IGFBP-5. CONCLUSION: In vivo expression of IGFBP-5 in the rat ovary is moderated by hormonal treatment both in terms of total expression and follicular localization.

Detection and in vivo hormonal regulation of rat ovarian type I and type II interleukin-I receptor mRNAs: increased expression during the periovulatory period.

OBJECTIVE: To study the expression, localization, and in vivo hormonal regulation of type I and type II interleukin-1 (IL-1) receptors in the rat ovary. METHODS: Segments of the cDNAs for rat type I and type II IL-1 receptors were cloned and used as probes in RNase protection assays and in situ hybridization. Tissues obtained from immature rats and hormonally treated rat ovaries were examined. RESULTS: Type I IL-1 receptor (IL-1R(1)) was ubiquitously expressed in rat tissues, including granulosa cells prepared from immature ovaries, whereas type II IL-1 receptor (IL-1R(2)) expression was restricted to macrophages, thymus, and lung. Hypophysectomy and subsequent treatment with FSH and/or diethylstilbestrol did not alter significantly the abundance of IL-1R(1) transcripts in the whole ovary. However, the relative amount of ovarian IL-1R(1) transcripts increased 7.3-fold 6 hours after the administration of hCG to pregnant mare serum gonadotropin-primed immature rats. During this time, IL-1R(1) mRNA was localized primarily in the granulosa cells. The increased expression of IL-1R(1) persisted 24 hours after hCG administration but declined to baseline by 48 hours. Ovarian expression of IL-1R(2) mRNA was observed only before ovulation in amounts that were approximately 70-fold lower than IL-1R(1). CONCLUSION: The increased intraovarian expression of IL-1R(1) in granulosa cells during the periovulatory period implies that this cell type has a heightened receptivity to IL-1 and provides further indirect evidence that this cytokine is involved in the ovulatory process.

Characterization and hormonal regulation of granulosa cell-derived insulin-like growth factor binding protein-4.

OBJECTIVE: Because of the potential importance of insulin-like growth factor binding protein-4 (IGFBP-4) to ovarian physiology and the obvious limitations imposed by in vivo-exclusive experimental paradigms, we set out to delineate the characteristics and hormonal regulation of granulosa cell-derived IGFBP-4 under in vitro circumstances. METHODS: Granulosa cells obtained by follicular puncture of the ovaries from diethylstilbestrol-primed intact immature rats were subjected to culture for up to 72 hours. Insulin-like growth factor binding protein-4 mRNA extracted from culture was subjected to Northern blot hybridization. Data normalization was assured by reprobing with the hamster Chinese hamster ovary B (CHOB) cDNA, and the IGFBP-4/CHOB ratio was calculated. Conditioned culture media were subjected to Western ligand blot before and after immunoprecipitation with a rat IGFBP-4-directed polyclonal antiserum (alpha-B104). RESULTS: Immunoprecipitation studies revealed granulosa cell-derived IGFBP-4 to be composed of a major 24-kDa species as well as a relatively minor 27-kDa moiety. Given cultures of untreated granulosa cells from immature estrogen-treated rats, transcripts corresponding to IGFBP-4 displayed an initial temporary decline culminating in a 6-hour nadir (a decrease of 67%; P < .05) followed by relatively prompt recovery (within 24 hours) to levels comparable to those noted at the outset of the culture (time 0). However, additional (albeit statistically insignificant) increments were noted at the 48-hour (but not 72-hour) time point. Treatment of granulosa cells with increasing concentration of FSH resulted in decrements of up to 30% (P < .05) in the steady-state levels of IGFBP-4 transcripts. A modest, biphasic, time-dependent response was noted for IGFBP-4 transcripts after treatment with high-dose FSH (100 ng/mL), an effect characterized by 24- and 48-hour increments (51% [P < .05] and 26% [P = .052] over untrated controls, respectively) and a 72-hour decrement (25%; P = .16). The concurrent provision of the C19 aromatase substrate androstenedione (10(-7) mol/L) to the culture medium from 72 hours enhanced the inhibitory effect of FSH (100 ng/mL) for a maximal decrement in IGFBP-4 transcripts of 49% (P < .05). Treatment with insulin-like growth factor (IGF)-I produced limited inhibition (up to 26%) of the steady-state levels of IGFBP-4 transcripts (P < .05). CONCLUSION: Findings indicate the existence of heterogeneously-sized IGFBP-4 species, of which the 27-kDa (as distinct from the 24-kDa) IGFBP-4 moiety constitutes a relatively minor component. The steady-state levels of granulosa cell-derived IGFBP-4 transcripts display relatively limited regulation in response to treatment with either FSH or IGF-I.

Rat ovarian insulin-like growth factor binding protein-4: a hormone-dependent granulosa cell-derived antigonadotropin.

OBJECTIVE: To assess the in vivo regulation of ovarian insulin-like growth factor binding protein-4 (IGFBP-4) mRNA expression by gonadotropins and estrogen. METHODS: Whole ovarian RNA, obtained from two models of follicular development, was extracted and analyzed by Northern blotting. Immature rats were treated with pregnant more serum gonadotropin (PMSG) followed 48 hours later with hCG, or alternatively were hypophysectomized and treated with FSH and/or diethylstilbestrol (DES). Localization of IGFBP-4 expression was assessed in the former study by in situ hybridization. Finally, the ability of human IGFBP-4 to antagonize FSH-stimulated progesterone accumulation was assessed in vitro. RESULTS: The ovarian content of IGFBP-4 transcripts increased threefold (P < .05) at 12 hours after PMSG but was near baseline at 24 and 48 hours. The abundance of IGFBP-4 mRNA increased (P < .05) again at 6 and 24 hours after hCG. The expression of IGFBP-4 was localized to granulosa cells of preantral (untreated) and small antral (12 hours after PMSG) follicles. No IGFBP-4 expression was noted in large (gonadotropin-primed) antral follicles. Hypophysectomy increased (P < .05) the ovarian content of IGFBP-4 mRNA by 1.5-fold, an effect further enhanced (1.8-fold; P < .05) by the provision of FSH and DES. In vitro studies revealed the ability of increasing concentrations (0.01-1 microgram/mL) of recombinant human IGFBP-4 to inhibit the FSH-supported accumulation of progesterone. CONCLUSION: Increased expression after administration of PMSG, hCG, and FSH/DES suggests that IGFBP-4 is a dynamic and hormonally responsive component of the ovarian cycle. The lack of expression in preovulatory follicles and its antigonadotropic actions in vitro imply that the attenuated expression of IGFBP-4 may constitute a requirement for successful follicular maturation.

Changes in the concentration of mRNAs for the inhibin subunits in ovarian follicles after administration of gonadotropins to progestin treated ewes.

RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.