A new antiestrogen, 2-(4-hydroxy-phenyl)-3-methyl-1-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-1H-indol-5-ol hydrochloride (ERA-923), inhibits the growth of tamoxifen-sensitive and -resistant tumors and is devoid of uterotropic effects in mice and rats.

PURPOSE: Tamoxifen is an antiestrogen used in women who have estrogen receptor (ER)-alpha-positive breast cancer. Unfortunately, resistance to tamoxifen is common in women with metastatic disease and side effects, including increased risk of endometrial cancer, exist. Here we describe the activity of a new selective ER modulator, ERA-923, in preclinical models focused on these limitations. EXPERIMENTAL DESIGN: The ability of ERA-923, 4-OH tamoxifen, or raloxifene to inhibit estrogen-stimulated growth was evaluated in cell-based and xenograft assays with tumor cells that are sensitive or resistant to tamoxifen. Uterine effects of selective ER modulators were compared in rodents. RESULTS: ERA-923 potently inhibits estrogen binding to ER-alpha (IC(50), 14 nM). In ER-alpha-positive human MCF-7 breast carcinoma cells, ERA-923 inhibits estrogen-stimulated growth (IC(50), 0.2 nM) associated with cytostasis. In vitro, a MCF-7 variant with inherent resistance to tamoxifen (10-fold) or 4-OH tamoxifen (>1000-fold) retains complete sensitivity to ERA-923. Partial sensitivity to ERA-923 exists in MCF-7 variants that have acquired profound tamoxifen resistance. In tumor-bearing animals, ERA-923 (10 mg/kg/day given p.o.) inhibits 17beta-estradiol-stimulated growth in human tumors derived from MCF-7, EnCa-101 endometrial, or BG-1 ovarian carcinoma cells, including a MCF-7-variant that is inherently resistant to tamoxifen. Raloxifene is inactive in the MCF-7 xenograft model. Unlike tamoxifen, droloxifene, or raloxifene, ERA-923 is not uterotropic in immature rats or ovariectomized mice. Consistent with this, tamoxifen, but not ERA-923, stimulates the growth of EnCa-101 tumors. CONCLUSIONS: In preclinical models, ERA-923 has an improved efficacy and safety compared with tamoxifen. Clinical trials with ERA-923 are in progress.

Mouse ascites golgi (MAG) mucin expression and regulation by progesterone in the rat uterus.

OBJECTIVE: To study the regulation of the blood group A-related high-molecular weight mucin glycoprotein epitope (mouse ascites golgi, MAG)-a menstrual cycle-dependent marker of endometrial receptivity-in a non-human endometrium model. METHODS: Immature Sprague-Dawley rats were injected with 1 microg of estradiol, 100 microg of testosterone, 100 microg of dexamethasone, 2.5 mg of progesterone (P), 0.325 mg of RU486, P and RU486, 100 microg of tamoxifen, or vehicle for 3 days, sacrificed, and the uteri were stained for MAG. Immunohistochemistry and blood analysis were the measurements used to compare the specimens from the exogenous hormonal and endogenous hormonal groups. Electron microscopy was used to locate the MAG epitope in one pseudopregnant adult Sprague-Dawley rat. RESULTS: The MAG epitope was present in endometrial glands of Sprague-Dawley rats, with maximal expression during proestrus and diestrus. Electron microscopy confirmed the Golgi location of this MAG epitope. In the untreated group, less than 0.5% of endometrial glands stained for MAG. The MAG was seen only in the glands of the P-treated rats and RU486 blunted this stimulatory effect by more than 95%. As little as 0.1 mg of P promoted MAG expression, with maximal response at 2.5 mg. Staining was seen 24 hours after P treatment, peaked at 72 hours, then declined. Induction of endogenous P by superovulation with pregnant mare serum gonadotropin (PMSG) and hCG (pseudopregnancy) also resulted in strong MAG glandular staining. CONCLUSION: Our results suggest that the MAG epitope is cyclically expressed and induced by P in rat endometrial glands.

Developing a SERM: stringent preclinical selection criteria leading to an acceptable candidate (WAY-140424) for clinical evaluation.

Estrogens are represented by a diverse group of compounds. Within this large family of molecules are tissue-selective estrogens that have been classified as selective estrogen receptor modulators (SERMs). These compounds are characterized by the fact that they exhibit both estrogen agonist and antagonist activity dependent upon the gene promoter and target tissue being examined. SERMs have been intensively studied over the past decade, especially since one, raloxifene, has been approved for the prevention and treatment of postmenopausal osteoporosis. While not a replacement for hormone replacement therapy (HRT), raloxifene can be an alternative to it and other treatments for osteoporosis. The ideal SERM would provide the positive benefits associated with HRT without the uterine and breast stimulation. Raloxifene does achieve some of the benefits of HRT, specifically on the skeleton and lipid metabolism with no apparent uterine effects, and a potential decreased risk of developing breast cancer associated with raloxifene therapy. However, there are a number of parameters that can be improved. A number of SERMs have been evaluated only to fail in development due to, for the most part, uterine safety issues. In order to develop an improved SERM, a stringent screening process was designed to select compounds that did not stimulate the uterus or breast. At the same time, these new compounds would have a positive impact on the skeleton and lipid metabolism with the additional improvement (over raloxifene) of a neutral effect on hot flashes. Under these strict conditions, WAY-140424 was developed and, to date, the preclinical pharmacology data have accurately predicted the clinical response demonstrated in phase I and II trials.

The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in HepG2 cells.

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-kappaB function. In the current study 17beta-estradiol-bound ERalpha interfered with cytokine-induced activation of a NF-kappaB reporter in HepG2 cells. The estrogen metabolite, 17alpha-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-kappaB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-kappaB interfered with the trans-activation properties of ERalpha. Ligand-bound ERalpha did not inhibit NF-kappaB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-kappaB transcriptional activity. Coexpression of the transcription coactivator CREB binding protein (CBP), but not steroid receptor coactivator 1a, reversed the ERalpha-mediated inhibition of NF-kappaB activity. Mammalian two-hybrid experiments also revealed that ligand-bound ERalpha can interact functionally with CBP-NF-kappaB complexes. We suggest that CBP targeting by ERalpha results in the inhibition of NF-kappaB and may occur through formation of transcriptionally inert multimeric complexes that are dependent upon the nature of the ERalpha ligand.

Recent insights into the varying activity of estrogens.

Recent advances have added substantially to our understanding of the biology of estrogens. Estrogens are no longer considered to differ only in potency. Two estrogens can have similar effects in one tissue and very different effects in another. Additionally, an estrogen can have different effects in different tissues. It is now recognized that there are at least two estrogen receptors, ER-alpha and ER-beta, and that it is quite likely that estrogens also work through non-genomic mechanisms. The development of new methods of chromatographic separation has aided substantially in our ability to characterize the composition of Premarin, including the identification of estradiene, the fourth-most abundant estrogen in Premarin. Recent studies have contributed to our understanding of the unique profile of Delta(8,9) dehydroestrone sulfate, another of the Premarin estrogens. It was found that Delta(8,9) dehydroestrone sulfate is an active estrogen with a distinct pharmacological profile that results in significant clinical activity in vasomotor, neuroendocrine and bone preservation parameters. However, it displayed little or no efficacy, at the dose studied, on other peripheral parameters normally affected by classical estrogens. Increasing knowledge of the unique profiles of the Premarin components, as well as their complex interaction, will help to increase our understanding of the clinical profile of Premarin.

Interacting quantitative trait loci control phenotypic variation in murine estradiol-regulated responses.

The steroid hormone estradiol (E2) elicits a spectrum of systemic and uterotropic responses in vivo. For example, E2 treatment of ovariectomized adult and sexually immature rodents leads to uterine leukocytic infiltration, cell proliferation, and organ growth. E2-regulated growth is also associated with a variety of normal and pathological phenotypes. Historically, the uterine growth response has been used as the key model to understand the molecular and biochemical mechanisms underlying E2-dependent growth. In this study, genome exclusion mapping identified two quantitative trait loci (QTL) in the mouse, Est2 and Est3 on chromosomes 5 and 11, respectively, that control the phenotypic variation in uterine wet weight. Both QTL are linked to a variety of E2-regulated genes, suggesting that they may represent loci within conserved gene complexes that play fundamental roles in mediating the effects of E2. Interaction and multiple trait analyses using the uterine leukocyte response and wet weight suggest that Est4, a QTL on chromosome 10, may encode an interacting factor that influences the quantitative variation in both responses. Our results show that E2-dependent responses can be genetically controlled and that a genetic basis may underlie the variation observed in many E2-dependent phenotypes.

A transcriptional coactivator, steroid receptor coactivator-3, selectively augments steroid receptor transcriptional activity.

Estrogen receptors ERalpha and ERbeta are members of the family of nuclear hormone receptors and act as ligand-inducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ERalpha and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ERbeta-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ERalpha interaction with SRC-3 was much higher than that observed for the ERbeta interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ERalpha and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ERalpha and ERbeta.

A novel human estrogen receptor beta: identification and functional analysis of additional N-terminal amino acids.

A novel human estrogen receptor beta (hERbeta) was cloned from human testis mRNA, ovary and thymus cDNA utilizing PCR and 5' RACE methods. The 5' end of hERbeta contained an additional open reading frame, in-frame and upstream of the published clones. hERbeta encodes a protein of 530 amino acids with an approximate molecular weight of 63 kDa and is larger than the previously reported rat, mouse and human protein. To determine the functional role of additional N-terminal amino acids, we compared the transcription functions of receptor lacking (hERbetaT) and receptor containing (hERbetaL) this N-terminal extension. hERbetaL is more active than hERbetaT in transactivating ERE-based reporter genes. hERbetaL, but not hERbetaT, attenuated cytokine mediated NFkappaB activation. Taken together, the additional N-terminal amino acids appear to play a role in modulating estrogen responsive gene expression in vitro.

Evidence for the genetic control of estradiol-regulated responses. Implications for variation in normal and pathological hormone-dependent phenotypes.

The ovarian steroid hormone estrogen (E2) elicits a multiplicity of both systemic and uterotropic responses in vivo. For example, the administration of E2 to ovariectomized (Ovx) and sexually immature rodents leads to uterine-specific inflammatory infiltrates. In this study, we quantitated the number of eosinophils and BM8+, Ia+, and CD4+ cells in uteri obtained from adult Ovx control and E2-treated C57BL/6J, C3H/HeJ, and (C57BL/6J x C3H/HeJ) (B6C3) F1 hybrid mice. All three strains exhibited a significant increase in the number of uterine eosinophils and BM8+ macrophages after E2 treatment. However, C57BL/6J and B6C3 F1 hybrid mice responded with a greater number of infiltrating eosinophils and macrophages as compared with C3H/HeJ. A similar analysis of Ia+ and CD4+ cells showed that E2 treatment either down-regulates or does not affect the number of such cells in all three strains. Genome exclusion mapping using a (C57BL/6J x C3H/HeJ) x C3H/HeJ backcross population localized Est1, the major locus controlling the number of eosinophils infiltrating the uterus after E2 treatment, to chromosome 4. In addition, suggestive linkage to marker loci on chromosomes 10 and 16 was detected and evidence for locus interaction is presented. Our results conclusively demonstrate that E2-regulated/ dependent responses can be genetically controlled, indicating that the phenotypic variation observed in both the normal and pathological effects of E2 may, in part, be due to a genetic component.

Chemotaxis of macrophages by a peritoneal fluid protein in women with endometriosis.

OBJECTIVE: To expand on a preliminary study comparing the chemotactic potential of peritoneal fluid (PF) from women with and without endometriosis and to characterize this activity via immunosuppressants and a protease. DESIGN: Case control study. SETTING: University center. PATIENT(S): Fifty-nine women with endometriosis and 44 without, undergoing laparoscopy. INTERVENTION(S): Collection of PF, endometriotic, ovarian, and endometrial biopsies at laparoscopy. MAIN OUTCOME MEASURE(S): Chemotactic activity of PF was tested via an in vitro assay alone and in the presence of immunosuppressants cyclosporin A (CSA), FK506, rapamycin, and type XVII-b(S-V8) protease and in media incubated with endometriotic, ovarian, or endometrial biopsy specimens. RESULT(S): The PF from women with endometriosis had significantly greater chemotactic activity (cells per well, mean +/- SD) than without endometriosis (142 +/- 39 versus 48 +/- 17). Cyclosporin A significantly inhibited the chemotactic activity of the endometriotic PF; FK506 and rapamycin did not. Incubation of media with endometriotic tissue, but not ovarian or endometrial, for > or = 7 hours displayed chemotactic activity. Protease type XVII-b(S-V8) added to endometriotic PF inhibited this chemotactic activity. CONCLUSION(S): Peritoneal fluid from patients with endometriosis contains a protein chemotactic factor attracting inflammatory cells into the peritoneal cavity, possibly secreted by endometriotic implants. This chemotactic factor may be a member of the immunophilin family because of its inhibition profile.

Estrogen receptor ligands modulate its interaction with DNA.

The estrogen receptor (ER) belongs to a superfamily of ligand-inducible transcription factors. Functions of these proteins (dimerization, DNA binding, and interaction with other transcription factors) are modulated by binding of their corresponding ligands. It is, however, controversial whether various ER ligands affect the receptor's ability to bind its specific DNA element (ERE). By using real time interaction analysis we have investigated the kinetics of human (h)ER binding to DNA in the absence and presence of 17beta-estradiol, 17alpha-ethynyl estradiol, analogs of tamoxifen, raloxifene, and ICI-182,780. We show that ligand binding dramatically influences the kinetics of hER interaction with specific DNA. We have found that binding of estradiol induces the rapid formation of a relatively unstable ER.ERE complex, and binding of ICI-182,780 leads to slow formation (ka is approximately 10 times lower) of a stable receptor-DNA complex (kd is almost 2 orders of magnitude lower). Therefore, binding of estradiol accelerates the frequency of receptor-DNA complex formation more than 50-fold, compared with unliganded ER, and more than 1000-fold compared with ER liganded with ICI-182,780. We hypothesize that a correlation exists between the rate of gene transcription and the frequency of receptor-DNA complex formation. We further show that a good correlation exists between the kinetics of hER-ERE interaction induced by a ligand and its biological effect.

Role of eosinophils in uterine responses to estrogen.

Administration of estradiol (E2) to ovariectomized mice results in a dramatic increase in uterine growth and an influx of eosinophilic leukocytes. This influx is mediated by stimulation of an E2-dependent eosinophilic chemotactic factor in the uterus (ECF-U). The role of this eosinophil infiltration in uterus is presently unknown but could involve early growth and/or remodeling processes. In an attempt to better define eosinophil function in uterine tissue, we produced ovariectomized mice severely depleted of circulating eosinophils by administration of a purified rat IgG monoclonal antibody against interleukin-5 (IL-5). Seven days later, animals were submitted to estradiol treatment. Experimental groups included mice treated with saline alone, saline followed by E2, IgG followed by E2, and anti-IL-5 followed by E2. Pretreatment with IL-5 antibodies led to no significant alteration in E2-induced increase in uterine wet weight. However, histological evaluation demonstrated a clear and almost complete blockade of E2-stimulated influx of eosinophils in anti-IL-5 treated animals. In addition, IL-5 antibody administration significantly reduced E2-induced increase in peroxidase activity. Dramatic reduction of eosinophils did not affect E2 stimulation of ECF-U activity by stromal cells or complement C3 synthesis by the epithelial cells. Thus, it appears that differences in E2 responses in eosinophil-deficient mice are not directly associated with presence or absence of eosinophils. Taken together, these data suggest that eosinophils most likely do not contribute to early growth in the E2-stimulated uterus. A possible role in other events such as remodeling remains to be elucidated.

Inflammatory changes of the endometrium in patients with minimal-to-moderate endometriosis.

OBJECTIVE: To investigate the endometrium of normal patients for chemotactic activity to neutrophils and macrophages and compare these findings with those of patients with endometriosis. STUDY DESIGN: Endometrial biopsies from patients with and without endometriosis were analyzed for chemotactic activity throughout the menstrual cycle. Glands and stroma of luteal samples were isolated to determine the source of this activity. Infiltrating cells to the endometrium were identified by immunohistochemistry with the use of the monoclonal antibody OKM1. RESULTS: Luteal endometrial samples from normal patients had higher chemotactic activity than samples from the proliferative phase; this was true for both cells types studied: macrophages, 84 versus 10 and neutrophils, 74 versus 11. Patients with endometriosis had high chemotactic activity in both proliferative and luteal biopsies: macrophages, 73 +/- 9 versus 78 +/- 1 and neutrophils, 41 +/- 18 versus 63 +/- 26. Stromal cells from luteal biopsies demonstrated a higher chemotactic activity than the epithelial component. Immunohistochemistry staining identified the infiltrating cells as macrophages. CONCLUSIONS: Extracts from endometrium of normal patients contain chemotactic activity for neutrophils and macrophages; this activity is higher in the secretory phase of the cycle. Endometriosis patients had high chemotactic activity throughout the menstrual cycle. Separation of the endometrial tissue into stroma and the glandular epithelium indicated that the stromal cells are the source of this factor.

Hormonal regulation of complement components and receptors throughout the menstrual cycle.

OBJECTIVES: Complement components have been recently demonstrated to be present in the reproductive tract. Among these components, C3 synthesis by glandular epithelium of the rat uterus has been shown to be regulated by estrogen; progesterone inhibits this synthesis. However, the hormonal regulation of C3 and the presence of other complement factors in the human has not been investigated to date. In this study we examined the presence and the hormonal regulation of different complement components and receptors in the human endometrium at various phases of the menstrual cycle of normally cycling women with no pelvic pathologic abnormalities. STUDY DESIGN: Endometrial tissue was obtained from normally cycling women, and immunohistochemistry was performed by means of monoclonal antibodies against C3, factors B, decay-accelerating factor, membrane cofactor protein, and complement receptor types 1, 2, and 3. The tissue was incubated with minimal essential media without methionine containing methionine labeled with sulfur 35. Immunoprecipitations were performed on the media with goat antihuman C3 antibody, and the proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: C3 was found to be present in the glandular epithelial cells of luteal endometrium. Biosynthesis as analyzed by immunoprecipitation with anti-C3 antibody was found to increase during the luteal phase of the cycle and to be minimal in the proliferative phase. Like C3, factor B and decay-accelerating factor were localized to the luteal glandular epithelial cells. In contrast, membrane cofactor protein was found to be present in the glandular epithelium throughout the menstrual cycle, and complement receptor type 1 was present only in the stromal compartment of luteal endometrium. Complement receptor type 3 was present only in the infiltrating leukocytes in the luteal endometrium, whereas complement receptor type 2 was undetectable. CONCLUSIONS: These findings suggest that several components of the complement system exist in the human endometrium in a hormone-dependent manner and may play a role in normal reproductive function.

Hormonal regulation of complement factor B in human endometrium.

PROBLEM: Recent investigations have demonstrated the presence of complement components in human endometrium in a cycle-specific manner. Luteal phase endometrium has been shown to synthesize complement C3 de novo, whereas proliferative endometrium produces little or no C3. Likewise factor B, which is critical to the activation of the alternative pathway of complement, has been shown to be present only in the glandular epithelium of luteal phase endometrium. This investigation was designed to determine if factor B is present in the endometrium in a high progesterone state such as pregnancy or with exogenous progesterone treatment. METHOD: Endometrial biopsies were obtained from patients on progesterone therapy. The endometrium of early pregnancy was evaluated by obtaining biopsies from patients with ectopic pregnancies as well as from patients undergoing therapeutic termination. Immunohistochemistry was performed on each biopsy using monoclonal antibodies to factor B. RESULTS: Our results demonstrate the presence of factor B in the glandular epithelial cells of the endometrium of patients treated with exogenous progesterone therapy. Additionally, factor B was localized to the glandular compartment of the endometrium from patients with ectopic gestations. Interestingly, the evaluation of an implantation site from an early gestation demonstrated factor B in the maternal decidua only; trophoblast did not exhibit the presence of factor B. CONCLUSION: Factor B exists in the endometrium in a hormone-dependent manner and is not expressed in fetal tissue in early gestation.

Antiandrogenic property of RU 486: enhancement of estrogen-induced uterine peroxidase activity in the rat.

The contragestational steroid RU 486 enhanced the increase in peroxidase activity produced by estradiol in estrogen-primed immature rat uteri and, like the antiandrogen flutamide, RU 486 reversed the inhibitory effect of testosterone on this estrogen-induced response. It antagonized the inhibition produced by progesterone but had no effect on peroxidase induction by itself or in unprimed immature animals. RU 486 also enhanced the effect of estradiol on the synthesis of complement component C3 in the rat uterus. The results confirm that RU 486 possesses antiandrogenic as well as antiprogestational properties. They also suggest that, in normal adult animals, the increase in peroxidase activity in the uterus in response to estrogen is not expressed fully but held in check by other endogenous steroids acting through their individual receptors.

Increased chemotactic activity of peritoneal fluid in patients with endometriosis.

OBJECTIVE: Our purpose was to investigate the ability of the peritoneal fluid of patients with endometriosis to induce chemotaxis of neutrophils and macrophages. STUDY DESIGN: Peritoneal fluid samples of patients with endometriosis (n = 20), normal fertile controls (n = 12), or patients with medical suppression (n = 8) were evaluated for chemotactic activity. Results of chemotactic activity were analyzed by analysis of variance. RESULTS: Peritoneal fluid of patients with endometriosis demonstrated a significantly higher chemotactic activity than that of patients without endometriosis or with medical suppression. Patients who had received medical treatment had the lowest chemotactic activity. (p < 0.001 for endometriosis vs control or treatment patients, p = 0.005 for control group vs treatment group). CONCLUSIONS: Patients with endometriosis have a higher chemotactic activity in their peritoneal fluid; prior medical treatment significantly reduces this activity. This chemotactic factor has an estimated weight of 20 kd. The nature and source of this chemotactic factor remains to be determined.

Progesterone and RU486 regulation of uterine complement C3 after prior induction with estradiol.

Previous results demonstrated that progesterone (P4) given simultaneously with estradiol (E2) prevented stimulation by E2 of complement C3 expression in the immature rat uterus. Northern blot analysis revealed that simultaneous administration of P4 was able to prevent the E2-stimulated increase in C3 mRNA concentration in the luminal epithelial cells. The purpose of the present investigation was to determine whether progesterone modulates C3 expression after the gene has been induced by prior administration of E2 and also to determine the reversibility of this effect by the concomitant administration of RU38486, 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]estra-4,9,-dien-3-one (RU486). This regulation was studied by examination of protein synthesis as well as mRNA concentrations. Immature 21-day-old female rats were treated with E2 for 2 days (1 microgram/day), followed 24 h later by P4 (500 micrograms) or vehicle. Uteri were removed 6, 9, and 18 h after progesterone treatment and the radiolabeled secreted proteins were analyzed by SDS-PAGE and immunoprecipitation using a goat anti-rat C3 antibody. In animals treated with vehicle, E2-stimulated C3 synthesis remained elevated at 6 and 9 h and returned to control values by 18 h. In contrast, the administration of P4 resulted in a decrease in C3 synthesis at 6 and 9 h with the greatest decrease observed at 9 h. Similar results were obtained when C3 mRNA concentrations were examined. E2-stimulated C3 mRNA concentrations were decreased in rats treated with progesterone compared to those treated with vehicle alone.2