Previous studies in the human suggest that the interleukin-1 (IL-1) system, may be an important paracrine/autocrine mediator in local intercellular interaction in endometrial tissue. In this study we have determined that IL-1 receptor type I (IL-1R tI) is expressed at the messenger RNA (mRNA) and protein levels in glandular cells and its ligand, IL-1 beta has been localized by immunohistochemical methods in endothelial cells and isolated stromal cells in the human endometrium throughout the menstrual cycle. IL-1R tI mRNA was detected in glandular epithelium using both specific complementary DNA and complementary RNA 32P-labeled probes. Human glandular epithelium contains a 5.1-kilobase mRNA transcript throughout the complete menstrual cycle. Quantitative densitometric analysis of slot blot hybridization signals shows an increase of IL-1R tI mRNA in both early and mid-late secretory phases in comparison with the proliferative phase (P < 0.05). IL-1R tI protein was localized in endometrial glandular epithelial cells using both indirect immunofluorescence and avidin-biotin-peroxidase methods. However, more intense staining for IL-1R tI was observed in lumenal epithelial cells compared with the staining present deep in the endometrial glands. Using the same methods, IL-1 beta was detected in endothelial cells of spiral vessels and isolated stromal cells throughout the menstrual cycle, and an increased staining from proliferative to secretory phase was observed. The detection of IL-1R tI in the human endometrial epithelium and its ligand, IL-1 beta, in isolated stromal cells and endothelial cells, is another example of possible communication between the immune and reproductive systems with special relevance to human implantation.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues.

The distribution of immunoreactive interleukin-1 receptor type I (IL-1R tI), IL-1 alpha, and IL-1 beta, and of macrophages, was investigated immunohistochemically in the mouse ovary during follicular growth, ovulation, and luteinization. For this purpose, an indirect immunofluorescence technique, using specific monoclonal antibodies against mouse IL-1R tI, mouse IL-1 alpha, IL-1 beta, and macrophage antigens (CD11b/CD18) was used with sections of paraffin-embedded ovaries from eCG and eCG/hCG-treated 12-wk-old B6C3F-1 female mice. During follicular development, IL-1 alpha, IL-1 beta, and IL-1R tI staining were confined to the theca-interstitial layer of growing follicles with one remarkable exception. Intense IL-1R tI still staining was present in the cytoplasm and plasma membrane of the murine oocyte. During ovulation, IL-1 alpha and IL-1 beta were still confined to the theca layer, but faint IL-1R tI staining was initiated in cumulus cells and in granulosa cells just before follicle rupture. Immediately after follicle rupture, granulosa cells stained positive for IL-1R tI, IL-1 alpha, and IL-1 beta. During luteinization, granulosa-luteal cells of the corpus luteum demonstrated strong IL-1R tI, IL-1 alpha, and IL-1 beta staining. Macrophages were detected in the theca layer and stroma, but never within the follicle before ovulation. Immediately after ovulation, there was a rapid entry of macrophages into the follicle, and macrophages were also present inside the corpus luteum. Our morphological results support a possible autocrine-paracrine role of the mouse ovarian IL-1 system in ovulation and luteinization.

We have investigated the relevance of interleukin-1 receptor type I (IL-1R tI) in the implantation process in vivo in a murine model. Indirect immunofluorescence experiments demonstrate that IL-1R tI is located in mouse endometrial lumenal epithelium with increased intensity in the periimplantation period, whereas IL-1 beta staining is located in the mouse placenta. PMSG/human CG (hCG)-stimulated and mated 12-week-old B6C3F-1 female mice were randomly allocated to three groups: A, control noninjected; B, buffer-injected animals; and C, animals injected ip with 20 micrograms recombinant human IL-1 receptor antagonist (rhIL-1ra) every 12 h beginning on pregnancy day 3. Injections were continued until day 9, and animals were killed 12 h after the last injection. Pregnancy rates in the three groups were: noninjected, 58.8% (10 of 17); buffer-injected, 73.7% (14 of 19); rhIL-1ra-injected, 6.7% (1 of 15), P = 0.0001155, Fisher exact test. To rule out the possibility that pregnancy failure was due to an embryotoxic effect of rhIL-1ra, 2-cell mouse embryos (n = 276) were flushed from the same group of animals used for in vivo experiments and cultured with increasing concentrations of rhIL-1ra: 0 microgram/ml (n = 91), 1 microgram/ml (n = 36), 50 micrograms/ml (n = 36), 100 micrograms/ml (n = 52), and 200 micrograms/ml (n = 61) rhIL-1ra. The percentages of 2-cell mouse embryos reaching the blastocyst stage after 72 h in culture were 85.7%, 91.6%, 94.4%, 96%, and 85.2%, respectively. We further cultured these blastocysts for 5 days on fibronectin-coated plates with or without 200 micrograms/ml rhIL-1ra. In both groups, hatching, attachment to fibronectin, outgrowth, and migration were documented to be similar. Furthermore, our longitudinal morphological study of embryonic implantation in control and rhIL-1ra-injected mice shows that the blockade of IL-1R tI interferes with the attachment of mouse blastocysts to maternal endometrium in vivo. In summary, we demonstrate that blockade of maternal endometrial IL-1R tI with IL-1ra prevents implantation in the mouse by interfering with embryonic attachment, without adverse effects on blastocyst formation, hatching, fibronectin attachment, outgrowth, and migration in vitro.

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.

Interleukin-1 receptor type I, IL-1 beta, IL-1 receptor antagonist, and human macrophages were immunohistochemically localized in the villous trophoblast, maternal-trophoblast interphase, and maternal decidua during early human implantation. Immunostaining for IL-1 receptor type I was present in the syncytiotrophoblast and hyperplastic endometrial glands in the maternal decidua. Immunoreactive IL-1 beta was present in the villous cytotrophoblast, syncytiotrophoblast, intermediate trophoblast, and maternal stromal decidual cells. IL-1 receptor antagonist staining was observed in the glandular endometrium of the maternal decidua and in isolated cells located inside the chorionic villi, intervillous space, and maternal decidua. Mature human macrophages, as defined by both CD/68+ and HAM56+, were present in the chorionic villi, maternal blood of intervillous space, and maternal decidua. Co-localization studies demonstrated that macrophages in all of the reported locations also stained for immunoreactive IL-1 beta. Our results show the shared presence in maternal and embryonic tissues of this receptor-agonist-antagonist system during early human implantation. This finding supports an autocrine/paracrine role for the IL-1 system in human implantation.

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The induction of LH receptors in granulosa cells is prerequisite for ovarian follicles to ovulate and form corpora lutea. Earlier studies have demonstrated the modulatory role of gonadotropins, growth factors, and GnRH on ovarian LH receptor content. We have now analyzed the influences of gonadotropins (FSH, LH, and PRL), several growth factors, and GnRH on LH receptor mRNA levels in cultured granulosa cells. Cells were obtained from immature estrogen-treated rats and cultured in medium containing FSH with or without growth factors or GnRH for 48 h. Some cells were also treated with FSH for 48 h, followed by treatment with FSH, LH, or PRL for another 2 days. Cellular total RNA was extracted, and blot hybridization with 32P-labeled LH receptor cRNA or 28S ribosomal RNA cDNA probes was performed. Treatment of granulosa cells with FSH increased the levels of five species of LH receptor mRNAs in a dose- and time-dependent manner. In FSH-primed cells, LH receptor mRNA levels were maintained by FSH, LH, and PRL. In contrast, treatment of cells with basic fibroblast growth factor or epidermal growth factor suppressed FSH induction of LH receptor mRNA in a dose-dependent manner, whereas treatment with insulin-like growth factor-I had no effect. In addition, GnRH suppressed FSH-stimulated LH receptor mRNA levels in a dose-dependent manner; the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting mediation by specific GnRH-binding sites. These studies demonstrated that the observed stimulatory effects of gonadotropins (FSH, LH, and PRL) and the inhibitory effects of growth factors (epidermal growth factor and basic fibroblast growth factor) and GnRH on LH receptor content are correlated to their regulation of LH receptor mRNA levels. The granulosa cell culture system should provide a useful model for studying LH receptor gene regulation.