Ovarian interleukin-1 receptor antagonist in rats: gene expression, cellular localization, cyclic variation, and hormonal regulation of a potential determinant of interleukin-1 action.

It is hypothesized that the intraovarian interleukin (IL)-1 system plays a prominent role in the regulation of follicular development and ovulation. A central component of the intraovarian IL-1 system is the IL-1 receptor antagonist (IL-1RA), a protein acting as a pure IL-1 receptor antagonist and one for which intracellular (icIL-1RA) and secretory (sIL-1RA) varieties have been described. It was the objective of this study to explore rat ovarian IL-1RA gene expression, to establish the identity and relative abundance of its alternative transcripts, to study its cellular localization, to determine its cyclic variation, and to assess its hormonal regulation. Protected IL-1RA cDNA fragments corresponding to either sIL-1RA or icIL-1RA were barely detectable in untreated whole ovarian tissue of immature rat origin. However, sIL-1RA transcripts reached a maximal value (3.3-fold increase over untreated control values; p < 0.05) 12 h after hCG administration (time of projected ovulation). In situ hybridization localized IL-1RA to mural, antral, and cumulus granulosa cells. Modestly intense staining was also apparent in oocytes. The basal pattern of sIL-1RA expression by cultured whole ovarian dispersates was characterized by a spontaneous increase to a peak value at 4 h. The early (4 h) sIL-1RA burst proved IL-1-, nitric oxide-, and protein biosynthesis-independent. However, treatment with IL-1beta led to a secondary sIL-1RA peak at 48 h, an effect that was substantially reversed by IL-1RA. This stimulatory effect of IL-1beta on IL-1RA expression proved relatively specific, and nitric oxide independent, but contingent upon de novo protein biosynthesis. The in vitro expression of icIL-1RA was barely detectable. Taken together, these in vivo and in vitro observations 1) document the rat ovary as a site of IL-1RA (sIL-1RA > cIL-1RA) expression, 2) localize the relevant transcripts to the granulosa cell, 3) disclose peak expression at the time of ovulation, and 4) establish IL-1 dependence.

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.

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.

The rat ovarian phospholipase A2 system: gene expression, cellular localization, activity characterization, and interleukin-1 dependence.

We have previously demonstrated that interleukin-1 beta (IL-1 beta), a putative intermediary in the ovulatory process, is a potent stimulator of ovarian PG biosynthesis. In this communication, we examine the possibility that this IL-1 effect reflects in part the induction of arachidonic acid mobilization by phospholipase A2 (PLA2). Molecular probing of whole ovarian material revealed the immature rat ovary to be a site of modest secretory PLA2 (sPLA2) gene expression. However, no change in ovarian sPLA2 gene expression was noted during the periovulatory period. Comparable probing for cytosolic PLA2 (cPLA2) failed to disclose a quantifiable signal. However, in situ hybridization localized both sPLA2 and cPLA2 (sPLA2 > cPLA2) transcripts to the granulosa cell layer of the ovarian follicle. Treatment of cultured whole ovarian dispersates with IL-1 beta produced significant (P < 0.01) increments in the steady state levels of transcripts corresponding to both sPLA2 (1.7-fold increase) and cPLA2 (5-fold increase), an effect reversed by an IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. Treatment with cycloheximide, a protein synthesis inhibitor, resulted in significant attenuation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Treatment with aminoguanidine, an inhibitor of inducible nitric oxide synthase, led to augmentation of the ability of IL-1 beta to up-regulate sPLA2 and cPLA2 gene expression as well as medium PLA2 activity. Total cellular PLA2 activity proved time, cell density, and calcium dependent, with an optimal pH of 8.0-9.0 and K(m) values in the low micromolar range (2-5 microM). Our observations 1) establish the rat ovary as a site of sPLA2 and cPLA2 gene expression, 2) localize the corresponding transcripts to the granulosa cell layer, and 3) establish IL-1 beta as an up-regulatory agent for ovarian sPLA2 and cPLA2 gene expression as well as for ovarian PLA2 activity. These findings also indicate that the IL-1 effect is 1) receptor mediated, 2) contingent in part upon de novo protein biosynthesis, and 3) inhibited by nitric oxide. These observations support the proposition that PLA2 may be a key component in the IL-1-stimulated biosynthesis of ovarian PGs.

Follicular atresia as an apoptotic process: atresia-associated increase in the ovarian expression of the putative apoptotic marker sulfated glycoprotein-2.

OBJECTIVE: To evaluate the possibility that morphologically confirmed/hypophysectomy-induced ovarian follicular atresia, a putative apoptotic process, is coupled to alterations in the steady-state levels of ovarian sulfated glycoprotein-2 (SGP-2) transcripts. METHODS: Hypophysectomy-induced follicular atresia in immature rats, morphologically confirmed at the light and electron microscopic levels, was correlated with alterations in the steady-state levels of ovarian SGP-2 transcripts as assessed by a solution hybridization/RNase protection assay. Cellular localization was accomplished by in situ hybridization technology. RESULTS: Hypophysectomy of the 24-day-old immature rat, an established precipitant of follicular atresia, led (3 days later) to a significant (P < .05) increase (up to 3.3-fold) in the relative abundance of densitometrically quantified ovarian SGP-2 transcripts compared with age-matched intact controls. Detailed time-course analysis after hypophysectomy revealed significantly (P < .05) increased ovarian SGP-2 mRNA expression as early as 2 days after hypophysectomy; no further increments were noted on days 4 or 8. Light microscopic analysis of comparable ovarian material 4 days after hypophysectomy revealed increased numbers of atretic follicles displaying large numbers of degenerating granulosa cells. Electron microscopic analysis of the degenerating cells of atretic follicles (from hypophysectomized rats) disclosed nuclear condensation and cytoplasmic shrinkage as well as apoptotic bodies at all levels of the granulosa cell layer. In situ hybridization established the granulosa cell of the intact untreated rat as the somatic cell concerned with SGP-2 gene expression. In turn, hypophysectomy led to an increase in SGP-2 expression at the level of the theca-interstitial cell, an effect prevented by the concurrent provision of pregnant mare serum gonadotropin (PMSG). The hypophysectomy-induced increase in ovarian SGP-2 transcripts was similarly reversed (54% inhibition by day 27) by the concomitant provision of FSH, an established antiatretic principle. The delayed administration (day 26) of a single dose of PMSG to rats hypophysectomized on day 24 eliminated the hypophysectomy-induced increase in ovarian SGP-2 transcripts as assessed on day 28. Qualitatively similar but quantitatively more pronounced increments in ovarian SGP-2 gene expression were obtained when atresia was induced by hypophysectomy of PMSG-primed immature rats. CONCLUSIONS: These observations establish the immature rat ovary as a site of SGP-2 gene expression and reveal hypophysectomy-induced follicular atresia to result in the up-regulation of ovarian (specifically, theca-interstitial) SGP-2 gene expression, an effect prevented by the concurrent provision of FSH or PMSG. To the extent that SGP-2 is an acceptable apoptotic marker, the present findings support the hypothesis that ovarian follicular atresia may be an apoptotic process.

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.