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In brief Changes in the endometrium prior to implantation may be critical in predicting pregnancy outcomes. This study shows that the endocrine system directs positional changes in CCR7+ cells before implantation, which may be critical for developing maternal tolerance. Abstract Suppression of the maternal immune system is vital for the implantation of the semi-allogeneic embryo. Although progress in understanding the dialogue between mother and embryo has been made, key interactions between maternal immune cells, hormones, and chemokines remain elusive. Uterine expression of the C-C chemokine receptor type 7 (CCR7) could recruit T regulatory cells and facilitate localized immune suppression. To test this concept, Ccr7 mRNA and protein were assessed in uterine tissue. Ccr7 mRNA expression peaked at day 4 in pregnant rat uteri and then declined at days 5 and 6. CCR7 protein showed similar quantitative changes. To test if female sex steroids affected the spatial distribution of CCR7-expressing cells, uteri from ovariectomized rats, progesterone-pretreated rats (2 mg daily), and progesterone-pretreated rats injected with estradiol (0.2 µg) were analyzed. Progesterone increased CCR7-positive (+) cells in the antimesometrial stroma. Progesterone and estradiol increased CCR7+ cells in the mesometrial stroma. Estradiol increased the density of cluster of differentiation 4 (CD4) positive cells in the mesometrial stromal region over progesterone alone. The density of cells expressing the T regulatory cell marker, forkhead box protein 3 (FOXP3), increased in the antimesometrial stroma in response to progesterone alone. Progesterone and estradiol increased FOXP3+ cells in the antimesometrial region of the stroma. Co-localization of CCR7, CD4, and FOXP3 in the stroma suggests CCR7+ cells are T regulatory cells. Polarization of CCR7+ cells in the endometrial stroma was an intrinsic response regulated by sex steroids and did not require the presence of an embryo.

The Kansas-IDeA Network of Biomedical Research Excellence (K-INBRE) is an infrastructure-building program funded by the National Institute of General Medical Sciences. Undergraduate education, through undergraduate research, is a key component of the program. The K-INBRE network includes 10 higher education institutions in Kansas and northern Oklahoma, with over 1,000 student participants in 16 yr. Since 2003, the K-INBRE has held an annual state-wide research symposium that includes national and regional speakers and provides a forum for undergraduates to give platform and poster presentations. The symposium is well attended by K-INBRE participants and has grown to a size of over 300 participants per year from all 10 K-INBRE schools. Two surveys were distributed to students and mentors to assess the impact of the symposium on student learning. Surveys (153) were distributed to students who participated in K-INBRE from 2013 through 2015 with a 51% response rate. Mentors were surveyed with a response of 111 surveys out of 161. Survey results indicate that students and mentors alike find the symposium to be beneficial and enriching of the student experience. Almost 80% of student respondents indicated that their participation in the symposium fostered appreciation of research. In short, the K-INBRE symposium provides a unique opportunity for students to gain experience in collecting, preparing, and communicating research in a professional environment. The collaborative experience of the annual K-INBRE symposium, the impact it has on student learning, and how it has influenced the research culture at our 10 institutions will be described.

The maternal uterus in mammals undergoes extensive remodeling in preparation for implantation of the semi-allogeneic embryo. Activation of the T cell homing receptor, CCR7, regulates multiple aspects of adaptive immunity. Genetic deletion of CCR7 reduces T regulatory (Treg) cell migration into mouse uteri and decreases embryo implantation. CCL19 and CCL21 are the sole ligands for CCR7. We hypothesized that CCL19 and/or CCL21 expression could attract Treg cells into the pre-implantation uterus and provide local immune suppression prior to implantation. Sprague Dawley rat uteri were removed from pregnant rats at Days 3-6. RNA was isolated and real time PCR was used to measure CCL19 and CCL21 expression. To determine the spatial distribution of these ligands, rat uteri were fixed, embedded in paraffin and uterine sections were analyzed by immunocytochemistry. The influence of sex steroids on the spatial distribution of CCL19 was evaluated in uterine sections from steroid treated rats. Hormonal control of CCL19 and CCL21 expression was investigated in a rat uterine stromal cell line treated with female sex steroids. Expression of CCL19 and CCL21 peaked at Day 4 of pregnancy compared with Days 3, 5 or 6 (Mann Whitney, p

Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3β (GSK-3β) and stimulating β-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidateWntgenes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments.Wnt5aincreased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. RelativeWnt5aexpression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increasedWnt5amRNA stability. Knockdown ofWnt5ain uterine stromal cell lines inhibited stromal cell proliferation and decreasedWnt5amRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease resulting from abnormal interactions between T and B cells. The acquisition of SLE is linked to genetic susceptibility, and diverse environmental agents can trigger disease onset in genetically susceptible individuals. However, the strongest risk factor for developing SLE is being female (9:1 female to male ratio). The female sex steroid, estradiol, working through its receptors, contributes to the gender bias in SLE although the mechanisms remain enigmatic. In a small clinical trial, monthly administration of the estrogen receptor (ERα) antagonist, ICI182,780 (fulvestrant), significantly reduced disease indicators in SLE patients. In order to identify changes that could account for improved disease status, the present study utilized fulvestrant (Faslodex) to block ERα action in cultured SLE T cells that were purified from blood samples collected from SLE patients (n = 18, median age 42 years) and healthy control females (n = 25, median age 46 years). The effects of ERα antagonism on estradiol-dependent gene expression and canonical signaling pathways were analyzed. Pathways that were significantly altered by addition of Faslodex included T helper (Th) cell differentiation, steroid receptor signaling [glucocorticoid receptor (GR), ESR1 (ERα)], ubiquitination, and sumoylation pathways. ERα protein expression was significantly lower (p 0.05) between SLE patients and controls. A previously undetected interaction between GR and ERα signaling pathways suggests posttranslational modification of steroid receptors in SLE T cells may alter ERα/GR actions and contribute to the strong gender bias of this autoimmune disorder.

Differentiation of uterine stromal cells is critical for the establishment of pregnancy. This study had two purposes: (i) to validate the use of the UIII rat uterine stromal cell model for investigating mechanisms underlying decidual cell differentiation, and (ii) to use this cell model to identify a molecular switch for cellular entry into the decidual cell differentiation pathway. Quiescent rat uterine stromal cells were transfected with a 500 bp segment of the decidual prolactin-related protein (dPRP) promoter ligated to a luciferase reporter gene. Cells were incubated in low-serum medium, or in low-serum medium containing progesterone (1 μM), estradiol 17-β (10 nM), cholera toxin (10 ng/ml) and interleukin-11 (10 ng/ml). Protein extracts were collected 48 h later and luciferase was measured in the cellular lysates. Cholera toxin and interleukin-11 stimulated luciferase expression (P< 0.05) and addition of sex steroids further increased (P< 0.05) dPRP promoter activity. Stromal cells did not proliferate (P< 0.05) under differentiation conditions. Deletion analysis of the dPRP promoter revealed maximal luciferase expression between −250 and −500 bp relative to the transcription start site. Comparison of cyclin E/Cdk2 activity between proliferating and differentiating cells showed a 3-fold increase (P< 0.05) at 12 h in differentiating cells. The results suggest that cyclin E/Cdk2 serves as a molecular switch for uterine stromal cell entry into the decidual cell differentiation pathway.

Progesterone pretreatment increases the number of synchronously proliferating stromal cells in the ovariectomized rat uterus, but estrogen is necessary to stimulate reentry into the cell cycle. To investigate the mechanisms underlying differential hormone actions, sexually mature ovariectomized rats were injected with progesterone (2 mg) for three consecutive days. Estradiol 17-beta (0.6 microg) was administered to initiate cell proliferation. Uterine samples were collected at timed intervals. Cell entry into DNA replication was monitored by injecting 5-bromo-2'-deoxyuridine (1 mg/100 g body weight) 2 h before necropsy. Demicolchicine (400 microg) was injected 30 min before necropsy to assess transit into M phase. Temporal progress through G1 was determined by spatial changes in cyclin D1/D3 proteins. Total cyclin D1/D3 protein and mRNA was measured by Western and Northern blotting. Estrogen increased the number of 5-bromo-2'-deoxyuridine-positive stromal cells (P0.05), compared with the number in rats treated with progesterone alone. An increase (P0.05) in the number of M-phase cells occurred at 12 h post estrogen. There was no evidence for epithelial cell proliferation in response to steroid treatments. Cyclin D1/D3 mRNA was expressed in the uteri of ovariectomized and hormone treated rats. The D-type cyclin proteins, however, were not evident in stromal cells without estrogen treatment. Progesterone pretreatment inhibited estrogen-dependent epithelial cell proliferation while redirecting D-type cyclin expression to the uterine stroma. Stromal cell transit through G1 required nongenomic steroid-dependent action on signal transduction pathways that control the nuclear localization and cell type-specific expression of the D-type cyclin proteins.

Progesterone is the only steroid hormone that is essential for the establishment and maintenance of pregnancy in all mammalian species that have been studied. Mice lacking the progesterone receptor (PR) by targeted mutagenesis exhibit abnormalities in all aspects of reproduction including sexual behavior, mammary gland development, ovulation, and implantation. Implantation in PR null mice fails, in part, because the uterine stromal cells cannot undergo differentiation (the decidual cell reaction). Uterine stromal cells do not divide without progesterone and proliferation is blocked by progesterone antibodies and PR antagonism. In spite of the preeminence of progesterone in female reproduction, its molecular mechanisms of action on target cell proliferation and differentiation are not well understood. Recent studies suggest that progesterone plays a direct role in regulating cell cycle transit by increasing the expression and activation of cell cycle regulatory complexes. Furthermore, this progesterone-dependent regulation of cell cycle transit may provide a unique window of opportunity for uterine stromal cells to exit the proliferative cycle, and if exposed to appropriate agents, enter into the differentiation pathway.

Cloning and sequencing of a cDNA amplified by RNA fingerprinting at the implantation site of pregnant rats revealed 80% similarity with Hβ58, previously shown to be essential for formation of the chorioallantoic placenta in the mouse. Hβ58 mRNA was detected in the endometrium of hormonally sensitized rats stimulated to undergo decidualization and in the contralateral uterine horns lacking a decidual stimulus, indicating that uterine expression of Hβ58 mRNA did not require decidualization or the presence of a blastocyst. Immunodetection in the early postimplantation uterus (Days 6–8 of pregnancy) showed Hβ58 localized in the luminal and glandular epithelia and some stromal cells. Decidual cells at Day 6 of pregnancy expressed Hβ58, and by Day 9 of pregnancy, the protein localized throughout the maternal decidua. The temporal and spatial distribution of Hβ58 in the developing chorioallantoic placenta was assessed at Days 10, 12, and 14 of pregnancy. Immunoreactive Hβ58 localized to erythroid cells ...

Previous experiments in our laboratory indicated that calcineurin expression and PP2B phosphatase activity increased when estrogen was cultured with SLE T cells but not with T cells from normal women. In this report we extended our findings to show that estrogen receptor (ER) antagonism by ICI 182,780 inhibited the estrogen-dependent increase in calcineurin mRNA and phosphatase PP2B activity indicating that estrogen action was mediated through the ER. Inhibition of de novo protein synthesis with cycloheximide suggested that the estrogen-dependent increase in T cell calcineurin mRNA was a direct effect of the ER and new protein synthesis was not required. Estrogen increased calcineurin mRNA in systemic lupus erythematosus (SLE) T cells at 6 h after the start of culture correlating with increased phosphatase activity at this same time. Phosphatase activity increased significantly (P0.02) in lupus T cells cultured for 8 h in estradiol-containing medium. Reverse transcription and polymerase chain amplification revealed that ER-beta and ER-alpha were expressed in female and male T cells from SLE patients and normal controls. However, calcineurin steady-state mRNA levels were unaffected by estradiol in cultured T cells from male SLE patients and normal male and female controls. These data indicate that estrogen, bound to the ER, evokes a direct increase in calcineurin expression in T cells from female lupus patients. This gender-specific response suggests that ER function is altered in women with the female predominant autoimmune disease, SLE.

Systemic lupus erythematosus (SLE) predominantly affects women (9:1 compared to men) of childbearing age and often decreases its intensity in postmenopausal women, suggesting that sex hormones play a role in its pathogenesis. Comparison of steady-state levels of calcineurin mRNA using RNase protection assays revealed increased calcineurin expression in response to estradiol in cultured T cells from nine female lupus patients. Calcineurin mRNA levels did not increase significantly in T cells from eight age-matched normal control female volunteers. Estrogen-dependent calcineurin mRNA increased in a dose-dependent fashion, while progesterone and dexamethasone did not increase calcineurin mRNA in patient cells. Lupus T cell calcineurin mRNA increased in response to estradiol at 6 h but not at 3 h. Calcineurin phosphatase activity increased in lupus T cell extracts after incubation of cells with estradiol, while phosphatase activity in normal T cells was unaffected by estrogen. Calcineurin expression in T cells from patients with vasculitis and rheumatoid arthritis taking medications similar to those taken by the lupus patients was unaffected by estradiol. This study provides the first evidence for a molecular marker of estrogen action in lupus patients and suggests that estrogen-dependent changes in lupus T cell calcineurin could alter proinflammatory cytokine gene regulation and T-B cell interactions.

Cell proliferation and differentiation in the rodent uterus are probably controlled by the interaction of female sex steroids with polypeptide growth factors. Uterine basic fibroblast growth factor (bFGF) mRNA was measured by RNase protection during the time (days 2–4) of endometrial cell proliferation in the pregnant rat. bFGF transcripts were detected at each of the 3 days of pregnancy examined. To investigate the influence of oestrogen and progesterone on bFGF mRNA accumulation, ovariectomized rats were treated with oestradiol for 48 h followed by a single injection of oestradiol, progesterone, the two steroids co-injected or oil vehicle alone. Uterine RNA was collected 6 h after the last hormone injection. Steroid treatments increased steady-state uterine bFGF mRNA compared with vehicle control animals as measured by RNase protection. Northern blot analysis of c-fos and c-jun mRNAs from these same treatment groups revealed increased protooncogene expression in the uterus of hormone treated rats compared with the control animals. Temporal analysis of bFGF mRNA in ovariectomized rats at 1, 3 and 6 h after acute oestrogen and oestrogen-progesterone co-administration showed a dual pattern of transcript accumulation. Both hormone treatments increased bFGF mRNA within 1 h compared with vehicle injected rats. Co-administration of the two hormones, however, repressed bFGF mRNA accumulation relative to oestrogen at 3 and 6 h. Together, these studies provide evidence that bFGF control of uterine cell proliferation in pregnant rats can occur from newly synthesized bFGF. Moreover, the results suggest that progesterone is a potent stimulator of bFGF expression in the uterus. Journal of Endocrinology (1997) 154, 75–84

Objective Systemic lupus erythematosus (SLE) is an autoimmune disease that affects women nine times more often than men. The present study investigates estradiol-dependent control of the calcium-buffering protein, calreticulin, to gain further insight into the molecular basis of abnormal T cell signaling in SLE T cells. Methods T cells were purified from blood samples obtained from healthy females and SLE patients. Calreticulin expression was quantified by real-time polymerase chain amplification. Calreticulin and estrogen receptor-α were co-precipitated and analyzed by Western blotting to determine if the proteins associate in T cells. Results Calreticulin expression increased ( p = 0.034) in activated control T cells, while estradiol decreased ( p = 0.044) calreticulin in resting T cells. Calreticulin expression decreased in activated SLE T cell samples and increased in approximately 50% of resting T cell samples. Plasma estradiol was similar ( p > 0.05) among SLE patients and control volunteers. Estrogen receptor-α and calreticulin co-precipitated from nuclear and cytoplasmic T cell compartments. Conclusions The results indicate that estradiol tightly regulates calreticulin expression in normal human T cells, and the dynamics are different between activated and resting T cells. The absence of this tight regulation in SLE T cells could contribute to abnormal T cell function.

Uterine stromal cells undergo mitosis and differentiate into the decidua just prior to the expected time of implantation in humans and rodents. We have utilized a culture system that will be suitable for study of the molecular mechanisms regulating stromal cell proliferation. Stromal cells were isolated from the uteri of ovariectomized rats and were cultured in chemically defined medium. Cultured cells express the mesenchymal markers vimentin and desmin. They do not express the epithelial marker cytokeratin. Serum-starved stromal cells were stimulated to proliferate in a time frame consistent with the cell cycle through addition of a panel of growth factors (basic fibroblast growth factor [bFGF], epidermal growth factor, platelet-derived growth factor, transforming growth factor alpha, insulin-like growth factor I) and hormones to the culture medium. None of the growth factors tested significantly stimulated proliferation in the absence of progesterone. Furthermore, progesterone was the only steroid of those tested that stimulated mitosis in the presence of growth factors. Stromal cell proliferation in response to progesterone and bFGF was dose dependent and saturable. Addition of the progesterone receptor antagonist mifepristone (RU486) and an inhibitor of tyrosine kinase receptor activation (suramin) abolished stromal cell mitosis. Progesterone receptors and fibroblast growth factor receptor 1 (FGFR1) were identified by immunoblot analysis in proliferating stromal cells. Taken together, these results show that cultured stromal cells maintain progesterone-dependent cell cycle control that is mediated via progesterone receptors. Moreover, the data indicate that bFGF control of stromal cell proliferation is modulated via a specific isoform of FGFR1 containing the three-loop immunoglobulin-like domain.

Recent studies suggest that steroid effects on uterine cell proliferation may be moderated by polypeptide growth factors. We now provide evidence that high affinity fibroblast growth factor (FGF) receptors are present temporally and spatially in the pregnant rat uterus (days 4-6) to support the idea that basic FGF action occurs via binding to its high affinity FGF receptor 1 (FGFR1). Reverse transcription-polymerase chain amplification indicates that both the full-length transcript and an alternatively spliced messenger RNA are present in the uterus. Western immunoblot analysis confirms that rat uterine membrane proteins contain two receptor isoforms, and these receptors bind basic FGF with high affinity and specificity. Immunolocalization of FGFR1 revealed receptor-positive cells in both the uterine stroma and epithelia on days 4-6 of pregnancy. However, the receptor was differentially localized in the disparate cell types. The nuclei of stromal cells were positive for FGFR1, whereas epithelial cell nuclei were negative. Together, these results suggest that FGF signal transduction in uterine stromal cells is mediated by activation of FGFR1.

Cellular proliferation and differentiation are critical components of uterine remodeling prior to embryonic implantation. Recent studies have shown that the ovarian hormones, estrogen and progesterone, modulate these cellular events through the production of growth factors. Basic fibroblast growth factor (bFGF) has been implicated in the control of cell proliferation, differentiation, and embryonic development. To clarify its role in uterine remodeling, the cellular distribution of bFGF was examined immunohistochemically in the rat uterus during early pregnancy (Days 2-6). Basic FGF localized intracellularly in stromal and epithelial cells and within the extracellular matrix at Days 2 and 3. It was distinctly evident at the apical surface of epithelial cells at Days 4 and 5 of pregnancy. Concurrent with this apical localization, bFGF was present in the uterine luminal fluid, suggesting release of this growth factor from epithelial cells. Embryonic implantation was accompanied by increased intracellular bFGF content in luminal epithelial and decidual cells. However, similar cells outside of the implantation site and in the artificially decidualized uterus did not express analogous bFGF levels, indicating that a unique signal from the embryo triggers bFGF expression. Changes in the cell-specific distribution of bFGF imply a multifunctional role for this growth factor in uterine cell proliferation, differentiation, and embryonic implantation. In addition, the apical release of bFGF from epithelial cells indicates utilization of a novel secretory pathway for bFGF export during early pregnancy.

Publisher Summary This chapter updates and adds new information to complement and extend previous reviews on gender differences in autoimmunity. It starts with general remarks on what is new in sex hormones and autoimmunity. It then discusses genes and sex differences with respect to autoimmune diseases, immune mediators, and signals that are under the influence of sex hormones, and updates the information on estrogen receptors (ER), both ERα and ERβ. The chapter discusses immune modulation following activation and/or blocking of estrogen receptors and the subsequent expression of the autoimmune state. In this chapter, recent work was selected that dealt with hormones and gender differences in autoimmunity. The interplay between sex hormones and the cellular–humoral–cytokine factors that could influence the expression of autoimmune diseases is discussed. Specific autoimmune diseases with female predominance are discussed with emphasis on systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren syndrome (SS), and neurologic diseases, particularly those with established animal models. Current and future applications of the basic information in the field of hormones and gender are outlined with emphasis on possible future therapeutic applications in autoimmune diseases with female predominance.

Fibronectin mRNA and protein content were examined during embryonic implantation in the rat uterus. Content of total fibronectin mRNA at day 6 of pregnancy increased relative to the non-pregnant uterus. In contrast, fibronectin protein content of the subepithelial stroma was relatively decreased except in the region directly surrounding the lumen, and this fibronectin immunoreactivity was sensitive to hyaluronidase treatment. These changes are likely to reflect the degradation and subsequent remodeling of the previously stable uterine extracellular matrix in preparation for embryonic implantation. A+,B-,V+ fibronectin mRNAs were present in both the non-pregnant and day 6 pregnant uterus with increased content of A+ and V+ fibronectin mRNAs in the latter. A+ fibronectin mRNA was distributed throughout the endometrial stroma of the non-pregnant uterus and content of the subepithelial stroma increased by day 4 of pregnancy, coincident with progesterone action on the endometrium. On day 6 of pregnancy, fibronectin mRNAs encoding the V95 and A regions were preferentially localized to the mesometrial zone of the subepithelial stroma. Accumulation of these mRNA splicing variants at the mesometrial zone was dependent upon decidualization, but the embryo was not required. Thus, there are two major changes in uterine fibronectin gene expression as a result of pregnancy: increased fibronectin mRNA content and mesometrial localization. These changes suggest a key function for fibronectin in implantation and imply the operation of a regulatory program of fibronectin gene expression which depends on hormonal sensitization and a nidatory stimulus. © 1992 Wiley-liss, Inc.

The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.

In a previous study specific protein binding to the uteroglobin (UG) promoter was detected in gel retardation assays using progesterone-dominated rabbit uterine nuclear extract proteins. Those findings have now been extended to reveal the components within that specific shifted band. Southwestern blotting and photoaffinity cross-linking of protein-DNA complexes by UV irradiation demonstrate binding of two proteins with apparent molecular masses of 94 and 115 kDa to a 126-basepair UG gene fragment (UG126-194/-68). To further investigate the tissue- and hormone-specific expression of the UG gene and to relate that specificity to promoter binding, RNA was prepared from rabbit uterus, kidney, and lung after 5 consecutive days of progesterone treatment. Northern blots of total RNA showed an absence of UG expression in kidney, while UG message was detected in uterus and lung. Protein binding to UG promoter DNA was absent in extracts from nuclei of kidney and HeLa cells, where the gene is not expressed, and from lung, where the gene is expressed but not regulated by progesterone. Digestion of the uterine protein-DNA complex using the nuclease activity of phenanthroline-copper ion and DNAase-I revealed two footprints. Protection was similar on both DNA strands, indicating no preference of protein binding to one DNA strand over the other. Taken together, the results provide strong indirect evidence that transcriptional activation of UG gene expression by progesterone requires binding of two additional proteins to UG promoter elements.

Systemic lupus erythematosus (SLE) is an autoimmune disease that occurs primarily in women of reproductive age. The disease is characterized by exaggerated T-cell activity and abnormal T-cell signalling. The mitogen-activated protein kinase (MAPK) pathway is involved in the maintenance of T-cell tolerance that fails in patients with SLE. Oestrogen is a female sex hormone that binds to nuclear receptors and alters the rate of gene transcription. Oestrogen can also act through the plasma membrane and rapidly stimulate second messengers including calcium flux and kinase activation. In this study, we investigated whether oestrogen influences the activation of MAPK signalling through the phosphorylation of extracellular signal–regulated kinase 1/2 (ERK1/2) in activated SLE T cells. SLE and control T cells were cultured in serum-free medium without and with oestradiol (10−7 M) for 18 h. The T cells were activated with phorbol 12 myristate 13-acetate and ionomycin for various time points (0–60 min), and the amount of phosphorylated ERK1/2 was measured by immunoblotting. There were no differences in ERK1/2 phosphorylation between SLE and control T cells at 5 and 15 min after the activation stimulus. However, comparison between the amount of phosphorylated ERK1/2 in SLE T cells from the same patients cultured without and with oestradiol showed a significant oestrogen-dependent suppression ( P = 0.48) of ERK1/2 in patients with inactive/mild systemic lupus erythematosus disease activity index (SLEDAI) (0–2) compared with patients with moderate (4–6) or active (8–12) SLEDAI scores. These results suggest that the suppression of MAPK through ERK1/2 phosphorylation is sensitive to oestradiol in patients with inactive or mild disease, but the sensitivity is not maintained when disease activity increases. Furthermore, studies are now necessary to understand the mechanisms by which oestrogen influences MAPK activation in SLE T cells.

Estrogen plays a role in the activation of systemic lupus erythematosus (SLE) and in upregulating intracellular signals by binding to the estrogen receptor(s). Fulvestrant (Faslodex, AstraZeneca Pharmaceuticals, Wilmington, DE, USA), an estrogen selective receptor downregulator, competes for receptor binding in vitro and inhibits estrogen action in target cells. We evaluated the efficacy, side effects, and expression of T cell activation markers, following the administration of fulvestrant or placebo to premenopausal patients with SLE.Twenty women with moderate SLE Disease Activity Index (SLEDAI; 7.87 +/- 3.7) were enrolled. They were premenopausal with regular menstrual cycles and not taking exogenous hormones. The study was double-blind and placebo-controlled. Ten patients received 250 mg fulvestrant intramuscularly for 12 months, and 10 received the placebo. All were observed monthly and 3 months after final fulvestrant/placebo injection. Measures studied were monthly SLEDAI scores, routine and serologic markers for lupus, and serum concentrations of estrogen and fulvestrant. Expression of T cell calcineurin and CD154 mRNA in peripheral T cells was measured by polymerase chain reaction. Medications the patients were taking were recorded each visit. Bone density was obtained at baseline and at visit 12.Sixteen patients completed the 15-month study, 8 from each group. SLEDAI improved significantly in the fulvestrant group at both 12 months (p = 0.02) and 15 months (p = 0.002), but serologic markers, routine laboratory tests, and bone density did not. Serum estrogen levels were higher in the fulvestrant group and dropped when fulvestrant was discontinued; these differences were not statistically significant. Medications for therapy of lupus to the fulvestrant group were reduced, whereas the placebo group medications were unchanged or increased. Comparison of relative values at individual timepoints revealed significantly lower median values for the T cell activation markers CD154 (p0.001) and calcineurin (p = 0.013) in the fulvestrant arm.Blocking estrogen receptors in vivo by an estrogen selective receptor downregulator could be considered as a new and relatively safe therapeutic approach in the management of SLE patients with moderately active disease for the 1-year study period.

Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in CD154 expression in T cells from females with systemic lupus erythematosus (SLE). This present study investigates if the estrogen-dependent increase in CD154 expression is due to stabilization of the messenger RNA. T cells from female SLE patients and controls were cultured for 18 h in serum-free medium without and with estradiol 17-β (107M). T cells were either unstimulated (resting) or were activated by further culture on anti-CD3 coated plates. Actinomycin D (25 σg/mL) was added to parallel cultures to inhibit new messenger RNA synthesis. CD154 messenger RNA stability was assessed by reverse-transcription polymerase chain amplification. Resting SLE ( n = 10, P = 0.88) and normal ( n = 7, P = 0.65) T cells showed no significant differences in message stability in response to estradiol. CD154 messenger RNA was also not significantly stabilized in activated SLE ( n = 10, P = 0.15) or activated normal ( n = 6, P = 0.077) T cells in response to estradiol. These findings indicate that the estrogen-dependent increase in CD154 in SLE T cells is not due to stability of the mRNA. These data are consistent with the postulate that estradiol stimulates CD154 transcription in SLE T cells.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-β. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-β (0.2 μg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3β (GSK-3β) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3β protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of β-catenin in these stromal cells. Translocation of β-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. β-Catenin binding to TCF/LEF increased (P

Systemic lupus erythematosus (SLE) is an autoimmune disease primarily affecting women. T cell activation markers (calcineurin, CD154) increase in SLE T cells cultured with estradiol 17-beta. Biological effects of estradiol are mediated through 2 receptor proteins, estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta). We compared the amount of estrogen receptor subtypes in T cells and measured the ability of receptor agonist-specific ligands to activate marker gene expression.T cells were isolated from 22 female patients with SLE and 17 control women. The amount of ER subtypes was measured by immunoblotting. Some T cells were cultured with ER-alpha or ER-beta-specific agonists. Receptor activation was measured by increased expression of the T cell activation markers CD154 and calcineurin.Although the amount of ER-alpha appeared to be less in SLE T cells than in control T cells, the difference was not statistically significant (p = 0.081). The quantity of ER-beta was similar in SLE and control T cells. The expression of ER-alpha or ER-beta was independent of menstrual cycle phase, age, or SLE disease activity. Calcineurin and CD154 expression increased in SLE T cells cultured in medium containing ER-alpha and ER-beta agonists.These data indicate that both ER subtypes activate calcineurin and CD154 in SLE but not in normal T cells. Variation in the amount of ER-alpha in SLE T cells suggests this receptor subtype participates in the sensitivity of SLE T cells to estrogen.

The female sex hormones estrogen and progesterone stimulate proliferation and differentiation of human and rodent uterine cells. The purpose of this chapter is to provide a method for isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth medium containing 10% fetal bovine serum. After several passages, the purity of the stromal cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle progression is assessed by flow cytometry, 3H-thymidine and BrdU incorporation, whereas proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by Northern and Western blotting whereas cyclin-cyclin-dependent kinase activity is monitored using immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported in this chapter provide a suitable model system to investigate the signal transduction events that stimulate hormone-dependent control of the cell cycle.

The female sex hormones estrogen and progesterone stimulate proliferation and differentiation of human and rodent uterine cells. The purpose of this chapter is to provide a method for isolating hormone-responsive rat uterine stromal cell lines that can be used to study steroid control of the cell cycle. Uteri from ovariectomized rats are differentially digested with trypsin to separate epithelial and stromal cells. The stromal cells are cultured in a standard growth medium containing 10% fetal bovine serum. After several passages, the purity of the stromal cell lines is determined using immunocytochemistry. Cell proliferation is studied by culturing the stromal cells in serum-free medium containing sex steroids and other mitogens. Cell cycle progression is assessed by flow cytometry, 3H-thymidine and BrdU incorporation, whereas proliferation is monitored using the MTT assay. Cell cycle regulators are visualized by Northern and Western blotting whereas cyclin-cyclin-dependent kinase activity is monitored using immune complex kinase assays. Uterine stromal cell lines isolated using the methods reported in this chapter provide a suitable model system to investigate the signal transduction events that stimulate hormone-dependent control of the cell cycle.

Evidence suggests that sex differences extend beyond the reproductive system and include sex-dependent differences in the cardiovascular, neural, and immune systems. This chapter reviews some differences in immune responses between males and females. In particular, this chapter focuses on the role of hormones as modulators of the immune response and their possible role in autoimmunity. Sex steroid hormone receptors have been identified in most cells comprising the immune system. However, the function of sex hormone receptors, working through their specific immune system gene targets, has not been widely explored. Systemic lupus erythematosus (SLE) is a gender-biased autoimmune disease with female predominance. The evidence for sex hormone influence on this disease is compelling, and new studies provide clues about the underlying mechanisms by which sex hormones may influence the development or progression of this prototypic autoimmune disease. Sex steroids have been shown to regulate the expression of genes that play critical roles in the adaptive immune response, including programmed cell death, cell proliferation, and signal transduction. Moreover, the evidence now emerging suggests that sex hormones can affect both lymphocyte development and influence mature lymphocytes in circulation. Sex differences that are relevant to human health and disease are not only dependent on sex but also are influenced by complex interactions between genetics and the environment. The future challenge is to develop a better understanding of the nature of these interactions and to identify the differences between the sexes that are consequential for human health.

Calcineurin is a key mediator of T cell activation. Previous studies in our laboratory showed a dose-dependent and hormone-specific increase in calcineurin expression in the T cells from females with systemic lupus erythematosus (SLE). This study investigates whether the estrogen-dependent increase in calcineurin expression is due to stabilization of the messenger RNA (mRNA).T cells from female patients with SLE and controls were cultured for 18 hours in a serum-free medium with and without estradiol-17 beta (10(-7) M). Some T cells were activated by further culture on anti-CD3-coated plates. Actinomycin D (25 micrograms/mL) was added to some cultures to inhibit new mRNA synthesis. Calcineurin mRNA stability was assessed by reverse-transcription polymerase chain amplification.Resting SLE (n = 9, P = .59) and normal (n = 5, P = .90) T cells showed no significant differences in mRNA stability in response to estradiol. Calcineurin mRNA was not significantly stabilized in activated SLE (n = 10, P = .12) or activated normal (n = 8, P = .09) T cells in response to estradiol. However, the amount of calcineurin mRNA stabilized in activated normal T cells (n = 8) was significantly greater (P = .02) compared with SLE T cells (n = 10) only after culture in medium without estradiol.These findings highlight the complex gene regulatory mechanisms underlying the differential action of estrogen on SLE T cells. Furthermore, the data indicate that increased calcineurin expression in SLE T cells is not due solely to estrogen-dependent stabilization of the message, and probably involves additional transcriptional regulatory mechanisms.

We have shown that estrogen receptor (ERa,ERb) transcripts are expressed in SLE and normal T cells. In this study, T cell nuclear extracts from female lupus patients and normal donors were tested for biologically active ER proteins capable of binding to the human estrogen response element (hERE) by electrophoretic mobility shift assays. When peripheral blood T cells were stimulated with 17b-estradiol (E2), PMA and ionomycin, two major retarded bands in T cell nuclear extracts exhibited a migration pattern similar to slow migrating protein-ERE complexes in human breast cancer cell extracts. T cells cultured only with E2 did not have these complexes. The formation of the complexes was inhibited by competition with the hERE cold oligonucleotide and partially with anti-ERa antibodies. There was no notable difference in the migration pattern of ERE-binding proteins between the SLE and normal T cell extracts. Together, these results suggest that activated human T cells, whether lupus-derived or normal-derived, contain biologically active ERa proteins. Other factors may be responsible for differential sensitivity of lupus T cells to estrogen.

Understanding of cell cycle regulation in hormonally responsive cells lags behind studies in other systems because few models have been available to identify the role of steroid hormones and their receptors in this process. This study investigates progesterone-dependent effects on the progression of normal uterine stromal cells through early G1 phase of the cell cycle. Quiescent rat uterine stromal cells were stimulated to reenter the cell cycle by adding serum-free medium containing medroxyprogesterone acetate (MPA) and basic fibroblast growth factor (FGF). [3H]thymidine incorporation increased significantly (P = 0.025) in cells stimulated with both FGF alone and MPA plus FGF compared with the control cells. Moreover, cells stimulated with MPA plus FGF incorporated significantly more (P = 0.01) [3H]thymidine than cells treated with FGF alone, suggesting requisite interactions between progesterone and FGF for stromal cell entry into S phase. Flow cytometric analysis of stimulated stromal cells showed FGF alone and MPA plus FGF increased significantly (P = 0.002) the percentage of cells in S phase at 12 h. Incorporation of bromodeoxyuridine into stromal cell nuclei indicated that FGF alone and MPA plus FGF increased the percentage of cells entering S phase at 18 and 24 h compared with the control cells. In addition, MPA plus FGF increased significantly (P = 0.001) the number of cells entering S phase at 24 h compared with FGF alone and sustained S phase entry compared with FGF alone, MPA alone, or the control cells. Stromal cells inhibited from G1 reentry by inhibition of mitosis showed accelerated entry into S phase in response to MPA plus FGF compared with FGF alone. Cyclin D1 messenger RNA increased in stromal cells treated with MPA plus FGF at 9, 12, and 15 h. Addition of RU 486 to cells stimulated with MPA plus FGF for 9 h reduced cyclin D1 messenger RNA accumulation by 40%. Western blot analysis of cyclin D1 immunoprecipitates indicated complex formation with both cyclin-dependent kinase 4 (Cdk4) and cyclin dependent kinase 6 (Cdk6). Cyclin D1-Cdk complexes and kinase activity correlated temporally with increased cyclin D1 expression in cells cultured with MPA plus FGF. Taken together, these results show that progesterone-FGF interactions increase cyclin D1 expression, correlating with accelerated stromal cell entry into S phase compared with cells treated with FGF alone. Moreover, progesterone plus FGF sustains the timing of stimulation for transit of uterine stromal cells through G1 into S phase compared with FGF alone.

Implantation of the mammalian embryo and development of the placenta are interactive processes that depend on two-way communication between genetically distinct individuals. Since the pioneering embryo transfer experiments of Chang (1), the importance of developmental synchrony between the uterus and embryo for successful implantation has been appreciated. Although the strictness of this relationship varies somewhat among species, some degree of maturity of both the embryo and the uterus is required for the initiation of implantation. An important correlate of uterine maturation in many species, including the human, is the proliferation and differentiation of uterine stromal cells just before implantation. This process is called decidualization. Decidualization is initiated prior to breachment of the basement membrane by the blastocyst, and decidual cells are in intimate contact with the embryo during its implantation into the uterine wall. As a normal part of placentation, some decidual cells subsequently undergo apoptosis to accommodate the growing conceptus.

Implantation of the mammalian embryo and development of the placenta are interactive processes that depend on two-way communication between genetically distinct individuals. On the maternal side, the hormonal status of the uterus is crucial to change the uterus from a hostile environment to one that is receptive to the blastocyst (1). On the embryonic side, development must proceed from the fertilized ovum to the blastocyst stage before an implantation reaction can be initiated. The importance of the synchrony between the uterus and embryo was originally suggested by the pioneering embryo transfer experiments of Chang (2). Although the strictness of this relationship varies somewhat among species, the requirement for developmental synchrony has now been demonstrated to some extent for all species studied (1,3,4). The establishment and maintenance of pregnancy are complicated processes that can be more easily conceptualized by subdividing prenatal life into three distinct phases. In the first phase, the embryo is free-living in the maternal reproductive tract. However, luminal fluids provide a rich source of growth factors and nutrients during this phase, particularly in ungulates (5). Preimplantation development is remarkably similar among species even though there is exceptional diversity in placental types occurring later in pregnancy (6). The second phase of prenatal life spans the period of attachment of the blastocyst to the uterus and subsequent development of the placenta and fetal organs. Embryonic loss at this stage is high (7,8) often because of inadequate development of the placental vascular connections (9). Failure to produce bidirectional signals in the appropriate temporal sequence or amount may be largely responsible for embryo losses at this time (reviewed in 10). The latter half of pregnancy is considered to be the third and final phase of prenatal development. During this time, the major changes involve growth of the fetus and the placenta (reviewed in 11).

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase II. Oocytes remain at metaphase II until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze-fracture analysis, and replicated oocyte cortical patches, we demonstrate stage-specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.