Nuclear Progesterone Receptor Research Articles

Gene knockout of nuclear progesterone receptor provides insights into the regulation of ovulation by LH signaling in zebrafish.

It is well established that the luteinizing hormone surge triggers ovulation, a dynamic process leading to the release of the mature oocyte from the ovarian follicle. But how this process controlled by LH signaling remains largely unknown in non-mammalian species. In this study, we investigated the roles of nuclear progesterone receptor (npr) in LH-induced ovulation. Our results indicate that the nuclear progesterone receptor serves as an important mediator of LH action on ovulation. This conclusion is based on the following results: (1) the expression level of npr peaks at the full-grown stage of the follicles; (2) the expression of npr is stimulated by LH signaling in vitro and in vivo; and (3) the npr null females are infertile due to ovulation defects. Moreover, we further show that LH signaling could induce ptger4b expression in an npr-dependent manner, and blockage of Ptger4b could also block hCG-induced ovulation. Collectively, our results not only demonstrate that npr serves an indispensable role in mediating the action of LH on ovulation in zebrafish, but also provide insights into the molecular mechanisms of the regulation of ovulation in fish.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

Molecular evidence of functional progesterone withdrawal in human myometrium.

Progesterone suppresses uterine contractility acting through its receptors (PRA/B). The mechanism by which human labour is initiated in the presence of elevated circulating progesterone has remained an enigma since Csapo first theorized of a functional withdrawal of progesterone in 1965. Here we report that in vitro progesterone-liganded nuclear PRB forms a complex including JUN/JUN homodimers and P54nrb/Sin3A/HDAC to repress transcription of the key labour gene, Cx43. In contrast, unliganded PRA paradoxically activates Cx43 transcription by interacting with FRA2/JUND heterodimers. Furthermore, we find that while nuclear progesterone receptor (PR) is liganded during human pregnancy, it becomes unliganded during both term and preterm labour as a result of increased expression of the progesterone-metabolizing enzyme 20α HSD and reduced nuclear progesterone levels. Our data provide a mechanism by which human labour can occur in the presence of elevated circulating progesterone and suggests non-metabolizable progestogen might represent an alternative new therapeutic approach to preterm birth prevention.

Progestin increases the expression of gonadotropins in pituitaries of male zebrafish.

Our previous study showed that the in vivo positive effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the major progestin in zebrafish, on early spermatogenesis was much stronger than the ex vivo ones, which may suggest an effect of DHP on the expression of gonadotropins. In our present study, we first observed that fshb and lhb mRNA levels in the pituitary of male adult zebrafish were greatly inhibited by 3 weeks exposure to 10nM estradiol (E2). However, an additional 24h 100nM DHP exposure not only reversed the E2-induced inhibition, but also significantly increased the expression of fshb and lhb mRNA. These stimulatory effects were also observed in male adult fish without E2 pretreatment, and a time course experiment showed that it took 24h for fshb and 12h for lhb to respond significantly. Because these stimulatory activities were partially antagonized by a nuclear progesterone receptor (Pgr) antagonist mifepristone, we generated a Pgr-knockout (pgr(-/-)) model using the TALEN technique. With and without DHP in vivo treatment, fshb and lhb mRNA levels of pgr(-/-) were significantly lower than those of pgr(+/+) Furthermore, ex vivo treatment of pituitary fragments of pgr(-/-) with DHP stimulated lhb, but not fshb mRNA expression. Results from double-colored fluorescent in situ hybridization showed that pgr mRNA was expressed only in fshb-expressing cells. Taken together, our results indicated that DHP participated in the regulation of neuroendocrine control of reproduction in male zebrafish, and exerted a Pgr-mediated direct stimulatory effect on fshb mRNA at pituitary level.

MP57-18 EXPRESSION OF STEROID HORMONE RECEPTORS IN RESIDUAL CANCER AND STROMAL CELLS AFTER NEOADJUVANT CHEMOHORMONAL THERAPY WITH DOCETAXAL FOR HIGH-RISK LOCALIZED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Localized: Surgical Therapy III1 Apr 2016MP57-18 EXPRESSION OF STEROID HORMONE RECEPTORS IN RESIDUAL CANCER AND STROMAL CELLS AFTER NEOADJUVANT CHEMOHORMONAL THERAPY WITH DOCETAXAL FOR HIGH-RISK LOCALIZED PROSTATE CANCER Shintaro Narita, Taketoshi Nara, Mingguo Huang, Koichiro Takayama, Kazuyuki Numakura, Hiroshi Tsuruta, Atsushi Maeno, Mitsuru Saito, Takamitsu Inoue, Norihiko Tsuchiya, Hiroshi Nanjo, Shigeru Satoh, and Tomonori Habuchi Shintaro NaritaShintaro Narita More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Mingguo HuangMingguo Huang More articles by this author , Koichiro TakayamaKoichiro Takayama More articles by this author , Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Atsushi MaenoAtsushi Maeno More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , Hiroshi NanjoHiroshi Nanjo More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.672AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies suggest that activation of steroid hormone receptors (SHRs) is associated with PCa progression and resistance to chemohormonal therapy. In this study, we investigated SHR expressions in human PCa tissues in patients who underwent radical prostatectomy (RP) with and without neoadjuvant chemohormonal therapy. METHODS A tissue microarray with 210 cores from 70 high-risk localized PCa patients who underwent RP without neoadjuvant therapy (NNA), with neoadjuvant hormonal therapy (NHT), or with neoadjuvant chemohormonal therapy with docetaxel (CHT) was used to assess the epithelial and stromal expressions of four SHRs including androgen receptor (AR), glucocorticoid receptor (GR), estrogen receptor (ER) alpha, and progesterone receptor (PR). Expression levels of SHRs between the three groups were statistically assessed. Multivariate analyses were performed to investigate the prognostic factors of BCR in patients who underwent surgery with CHT. RESULTS AR and GR were expressed in both residual cancer and stromal cells, whereas ER alpha and PR were mainly expressed in stromal cells. The mean nuclear AR immunoreactivity score in residual cancer cells was significantly higher in the NNA group than in the NHT and CHT groups. The mean nuclear GR immunoreactivity score in residual cancer cells and mean nuclear ER alpha and PR in stromal cells were significantly higher in the CHT group than in the NNA and NHT groups. The patients with a low PR immunoreactivity score in stromal cells had a significantly higher BCR rate than those with a high nuclear immunoreactivity score (p = 0.011). In multivariate analysis, the number of risk factors constituting the classification of high-risk, a preoperative PSA level, pathological chemotherapy effect, and low nuclear PR immunoreactivity score in stromal cells were independent prognostic factors for BCR. CONCLUSIONS Stromal PR expression may be a potential prognostic marker for BCR in high-risk PCa patients who underwent radical prostatectomy after neoadjuvant chemohormonal therapy. The expression of SHRs in both residual cancer and stromal cells may play an important role in PCa resistance to chemohormonal therapy. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e759-e760 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Shintaro Narita More articles by this author Taketoshi Nara More articles by this author Mingguo Huang More articles by this author Koichiro Takayama More articles by this author Kazuyuki Numakura More articles by this author Hiroshi Tsuruta More articles by this author Atsushi Maeno More articles by this author Mitsuru Saito More articles by this author Takamitsu Inoue More articles by this author Norihiko Tsuchiya More articles by this author Hiroshi Nanjo More articles by this author Shigeru Satoh More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Knockdown of Progesterone Receptor (PGR) in Macaque Granulosa Cells Disrupts Ovulation and Progesterone Production.

Adenoviral vectors (vectors) expressing short-hairpin RNAs complementary to macaque nuclear progesterone (P) receptor PGR mRNA (shPGR) or a nontargeting scrambled control (shScram) were used to determine the role PGR plays in ovulation/luteinization in rhesus monkeys. Nonluteinized granulosa cells collected from monkeys (n = 4) undergoing controlled ovarian stimulation protocols were exposed to either shPGR, shScram, or no virus for 24 h; human chorionic gonadotropin (hCG) was then added to half of the wells to induce luteinization (luteinized granulosa cells [LGCs]; n = 4–6 wells/treatment/monkey). Cells/media were collected 48, 72, and 120 h postvector for evaluation of PGR mRNA and P levels. Addition of hCG increased (P < 0.05) PGR mRNA and medium P levels in controls. However, a time-dependent decline (P < 0.05) in PGR mRNA and P occurred in shPGR vector groups. Injection of shPGR, but not shScram, vector into the preovulatory follicle 20 h before hCG administration during controlled ovulation protocols prevented follicle rupture in five of six monkeys as determined by laparoscopic evaluation, with a trapped oocyte confirmed in three of four follicles of excised ovaries. Injection of shPGR also prevented the rise in serum P levels following the hCG bolus compared to shScram (P < 0.05). Nuclear PGR immunostaining was undetectable in granulosa cells from shPGR-injected follicles, compared to intense staining in shScram controls. Thus, the nuclear PGR appears to mediate P action in the dominant follicle promoting ovulation in primates. In vitro and in vivo effects of PGR knockdown in LGCs also support the hypothesis that P enhances its own synthesis in the primate corpus luteum by promoting luteinization.

Abstract P3-09-03: Combined effects of soy isoflavones and a high fat diet on the mammary gland in an animal model of diet-induced obesity

Abstract Scope: Obesity is a major risk factor for the development of breast cancer whereas isoflavone (ISO) exposure is discussed to reduce this risk. Aim of this study was to investigate effects of dietary soy ISO intake on proliferation and estrogenicity of ISO in the mammary gland of obese female Wistar rats. Methods: Female Wistar rats (5 – 7 rats / group) grew up on low fat ISO-depleted diet (LF IDD) or ISO-rich diet enriched with a soy based commercial extract (LF IRD; ISO: 467 mg / kg diet). Starting postnatal day 83, ovariectomized (OVX) and intact animals received high fat diet for 12 weeks to induce obesity in the absence (HF IDD) or presence of ISO (HF IRD, ISO: 431 mg / kg diet). A special diet switch group (HF IRD switch OVX), grew up on LF IDD but switched to HF IRD after ovariectomy. This mimics the short term exposure to ISO in postmenopausal Western women who take ISO supplements. Two groups receiving LF diet either with or without ISO (LF IRD, LF IDD) lifelong served as control. From ablactation until the end of the experiment body weight and food consumption were monitored twice a week. After 12 weeks of HF diet animals were sacrificed. Body weight, visceral fat mass, and serum leptin were measured, and breast tissue was excised. Protein expression of proliferating cell nuclear antigen (PCNA) and progesterone receptor (PR) in breast tissue was analyzed by both immunohistochemistry (IHC) and Western Blot as markers for proliferation and estrogenicity of ISO, respectively. Results: Analysis of ISO plasma levels revealed 1400 nM in LF IRD group and 300 – 700 nM in HF IRD groups. HF diet increased body weight, visceral fat mass and serum leptin levels compared to LF diet. In the mammary gland HF increased expression of proliferation marker PCNA and PR as compared to LF groups. Lifelong but not short term (HF IRD switch OVX) ISO exposure reduced body weight, visceral fat mass and leptin levels in HF OVX rats. In the mammary gland lifelong ISO exposure reduced PCNA expression in both LF and HF intact animals whereas in HF OVX animals lifelong ISO exposure increased PCNA expression compared to short term ISO exposure. PR expression increased in HF IRD OVX compared to HF IDD OVX. Conclusion: The ISO plasma levels of the rats are comparable to the average ISO plasma levels as found in Asian population. Our results show that lifelong ISO intake reduces the risk to develop obesity in female rats. In the mammary gland lifelong ISO exposure decreases cell proliferation and shows estrogenicity by increasing PR expression. Effects of short term ISO exposure are less strong compared to lifelong ISO exposure. This supports the hypothesis that only lifelong but not short term ISO exposure can reduce the risk to develop breast cancer. Citation Format: Kurrat A, Diel P, Blei T, Kluxen F, Mueller D, Pichotta M, Soukup S, Kulling S, Oden C. Combined effects of soy isoflavones and a high fat diet on the mammary gland in an animal model of diet-induced obesity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-09-03.

Abstract P1-01-16: Performance evaluation of two ready-to-use antibodies under development for the Dako Omnis automated staining platform on breast carcinoma specimens: Anti-estrogen receptor α clone EP1 and anti-progesterone receptor clone PgR 1294

Abstract Background: The expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) in breast carcinomas is a strong predictor of the efficacy of hormonal therapy for breast cancer patients as well as providing a degree of prognostic information. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) configured as FLEX ready-to-use antibodies have been tested on the Dako Omnis automated staining platform. These products are in performance evaluation and are not commercially available. A series of concordance studies were performed to evaluate the performance characteristics of these monoclonal antibodies on breast cancer tissue specimens: anti-ERα clone EP1/Dako Omnis was compared to (a) anti-ERα clone EP1/Autostainer Link 48 (238 specimens) and to (b) anti-ERα clone SP1/Autostainer (116 specimens), and anti-PR clone PgR 1294/Dako Omnis was compared to (a) anti-PR clone PgR 636/Autostainer Link 48 (289 specimens) and to (b) anti-PR clone 16 (Leica Biosystems, Newcastle, UK) (144 specimens). In addition, the specificity of the ER and PR antibodies for Dako Omnis was evaluated on a set of normal tissue specimens. Methods: Formalin-fixed, paraffin-embedded (FFPE) human breast carcinoma specimens and normal tissues were obtained from commercial providers or local hospitals. The specimens had no associated personal information and were not traceable back to the tissue donors. Tissue pretreatment and immunohistochemical staining were performed using the recommended protocol for each antibody and staining platform. The stained slides were evaluated for nuclear ER or PR expression according to ASCO/CAP guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. The concordance studies included breast cancer specimens covering the clinical range of ER or PR expression with approximately half the specimens in the negative (&amp;lt;1%) category, and at least 10% of the specimens in the weakly positive (≥1 ≤10%) category in each study. Two-sided Wilson Score 95% Confidence Intervals were calculated using JMP software (SAS Institute, USA). For the analytical specificity studies the presence or absence of specific staining in the various normal tissue types was recorded. Results: High concordance rates were observed with both anti-ERα clone EP1/Dako Omnis and anti-PR clone PgR 1294/Dako Omnis compared to the other ER/PR antibodies, with overall agreement rates exceeding 95% in all of the comparative studies. On a set of normal tissues, specific positive nuclear staining was observed only in tissue types known to express ERα or PR. Conclusions: Monoclonal antibodies anti-ERα clone EP1 and anti-PR clone PgR 1294 configured as FLEX ready-to-use on Dako Omnis are sensitive and specific assays for detecting estrogen receptor and progesterone receptor in FFPE tissues. In comparison testing for assessment of hormonal receptor status on breast carcinoma specimens, anti-ERα clone EP1/Dako Omnis and anti-PR clone PgR 1294/Dako Omnis were highly concordant with commercially-available ER or PR antibodies. Citation Format: Viale G, Dell'Orto P, Falzon M, Fält A, Hicks D, Hoff K, Jakobsen K, Jensen LB, Levy YY, McMahon L, Miller K, Russo L. Performance evaluation of two ready-to-use antibodies under development for the Dako Omnis automated staining platform on breast carcinoma specimens: Anti-estrogen receptor α clone EP1 and anti-progesterone receptor clone PgR 1294. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-16.

Expression of Progesterone Receptor Membrane Component 1 (PGRMC1), Progestin and AdipoQ Receptor 7 (PAQPR7), and Plasminogen Activator Inhibitor 1 RNA-Binding Protein (PAIRBP1) in Glioma Spheroids In Vitro.

Objective. Some effects of progesterone on glioma cells can be explained through the slow, genomic mediated response via nuclear receptors; the other effects suggest potential role of a fast, nongenomic action mediated by membrane-associated progesterone receptors. Methods. The effects of progesterone treatment on the expression levels of progesterone receptor membrane component 1 (PGRMC1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), and progestin and adipoQ receptor 7 (PAQR7) on both mRNA and protein levels were investigated in spheroids derived from human glioma cell lines U-87 MG and LN-229. Results. The only significant alteration at the transcript level was the decrease in PGRMC1 mRNA observed in LN-229 spheroids treated with 30 ng/mL of progesterone. No visible alterations at the protein levels were observed using immunohistochemical analysis. Stimulation of U-87 MG spheroids resulted in an increase of PGRMC1 but a decrease of PAIRBP1 protein. Double immunofluorescent detection of PGRMC1 and PAIRBP1 identified the two proteins to be partially colocalized in the cells. Western blot analysis revealed the expected bands for PGRMC1 and PAIRBP1, whereas two bands were detected for PAQR7. Conclusion. The progesterone action is supposed to be mediated via membrane-associated progesterone receptors as the nuclear progesterone receptor was absent in tested spheroids.

Differences in progesterone concentrations and mRNA expressions of progesterone receptors in bovine endometrial tissue between the uterine horns ipsilateral and contralateral to the corpus luteum.

Because the establishment of pregnancy begins at the uterine horn ipsilateral to the corpus luteum (ipsi-horn) in cattle, levels of progesterone (P4) and receptor expression in the endometrial tissue, which regulate the intrauterine environment for embryo development, may differ between the ipsi-horn and the uterine horn contralateral to corpus luteum (contra-horn). The aim of the present study was to determine the endometrial tissue P4 concentrations and nuclear progesterone receptor (PGR), progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 mRNA expressions in the cranial and middle parts of the uterine horns during the luteal phase. The results showed higher endometrial tissue P4 concentrations in the cranial part of the ipsi-horn than in that of the contra-horn (P<0.01); however, no change in the endometrial tissue P4 concentrations was evident during the luteal phase. The PGR mRNA expression was higher during the early luteal phase (P<0.05), but no differences between the horns were evident. However, PGRMC1 mRNA expression during the early luteal phase was higher in the cranial part of the ipsi-horn than in that of the contra-horn (P<0.05). In the middle part, there were no changes in the endometrial tissue P4 concentrations and P4 receptor expressions during the luteal phase. In conclusion, the differences in dynamics of endometrial tissue P4 concentrations and P4 receptor expressions between the uterine horns ipsilateral and contralateral to the ovary containing a corpus luteum may cause differences in the intrauterine environment for both the ipsi- and contra-horns.

Expression and immunolocalization of membrane progesterone receptors in the bovine oviduct

The oviduct plays a crucial role in the transport and maturation of gametes and ensures suitable conditions for fertility and early embryo development. One regulator of oviduct function is progesterone (P4), which affects the cell by interacting with nuclear progesterone receptors (PGRs) and through nongenomic mechanisms, presumably involving membrane PGRs. The aim of this study was to evaluate the expression of messenger RNAS (mRNAs) and proteins for progesterone receptor membrane component (PGRMC) 1 and 2 and membrane progestin receptors (mPR) α, β, and γ and to use immunohistochemistry to demonstrate their cell-specific localization in the bovine oviduct. Oviducts ipsilateral and contralateral to the corpus luteum or to the dominant follicle were collected from cows on days 6 to 12 (midluteal stage) and 18 to 20 (follicular stage) of the estrous cycle and divided into 3 parts (infundibulum, ampulla, and isthmus). There were no differences (P > 0.05) in the PGRMC1, PGRMC2, mPRα, β, and γ mRNA expression between ipsi- and contralateral oviducts. However, the same parts of the oviduct collected during the different stages of the estrous cycle showed higher (P < 0.05) mRNA levels of PGRMC1, PGRMC2, and mPRα on days 18 to 20 than on days 6 to 12 of the estrous cycle. mPRα and mPRβ mRNA levels were higher (P < 0.05) in the infundibulum than in the isthmus, whereas PGRMC1 expression was higher (P < 0.05) in the infundibulum than in ampulla. Immunohistochemistry was used to detect PGRMC1, PGRMC2, PRα, β, and γ proteins in all parts of both oviducts from days 6 to 12 and 18 to 20 of the estrous cycle. There were no differences in the staining intensity and cellular localization of the studied proteins between the ipsi- and contralateral oviducts and between the studied stages of the estrous cycle. A strong positive reaction was observed in luminal cells, but this reaction was less evident in myocytes and stromal cells. All proteins were also localized to the endothelial cells of blood vessels. These results suggest that membrane progesterone receptors, may be involved in the regulation of oviduct motility, secretory function, and blood flow in this organ.

Immunohistochemical Performance of Estrogen and Progesterone Receptor Antibodies on the Dako Omnis Staining Platform: Evaluation in Multicenter Studies.

The analysis of estrogen receptor (ER) and progesterone receptor (PR) expression levels by immunohistochemistry is an important part of the initial evaluation of breast cancer and critically important in treatment planning. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) antibodies are in development for the Dako Omnis automated staining platform. These antibodies are not yet commercially available and are in performance evaluation, including the 4 international, multicenter studies reported here. For each antibody, a reproducibility study and a method comparison study was done in a randomized manner in order to test the antibodies under conditions closest to real-world user conditions. The reproducibility studies included 5 staining runs on the Dako Omnis with 20 formalin-fixed and paraffin-embedded human breast carcinoma specimens in 3 independent laboratories, and the method comparison studies included several hundred specimens stained on the Dako Omnis and on the Autostainer Link 48 platforms. Stained slides were evaluated for nuclear ER or PR expression according to American Society of Clinical Oncology/College of American Pathologists guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. For both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis, high reproducibility agreement rates were obtained on the interrun, interlaboratory, and interobserver endpoints. High concordance rates were observed between the specimens stained on the Dako Omnis platform and the Autostainer Link 48 platform. Staining quality was excellent for both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis. These results suggest that these antibodies are reliable and reproducible tools for immunohistochemistry analysis of ER and PR expression levels in formalin-fixed and paraffin-embedded breast carcinoma tissues on the Dako Omnis platform.

Exposure to the Synthetic Progestin, 17α-Hydroxyprogesterone Caproate During Development Impairs Cognitive Flexibility in Adulthood

The synthetic progestin, 17α-hydroxyprogesterone caproate, is increasingly used for the prevention of premature birth in at-risk women, despite little understanding of the potential effects on the developing brain. Rodent models suggest that many regions of the developing brain are sensitive to progestins, including the mesocortical dopamine pathway, a neural circuit important for complex cognitive behaviors later in life. Nuclear progesterone receptor is expressed during perinatal development in dopaminergic cells of the ventral tegmental area that project to the medial prefrontal cortex. Progesterone receptor is also expressed in the subplate and in pyramidal cell layers II/III of medial prefrontal cortex during periods of dopaminergic synaptogenesis. In the present study, exposure to 17α-hydroxyprogesterone caproate during development of the mesocortical dopamine pathway in rats altered dopaminergic innervation of the prelimbic prefrontal cortex and impaired cognitive flexibility with increased perseveration later in life, perhaps to a greater extent in males. These studies provide evidence for developmental neurobehavioral effects of a drug in widespread clinical use and highlight the need for a reevaluation of the benefits and potential outcomes of prophylactic progestin administration for the prevention of premature delivery.

Expression and localization of progesterone receptor membrane component 1 (PGRMC1) in the rhesus macaque cervix

Progestogens can have rapid actions on cervix epithelium to alter mucus secretion (1). PGRMC1 is a member of a group of progesterone binding proteins that may mediate rapid non-classical progesterone (P) effects in target cells (2). PGRMC1 could also mediate the action of P in cells that lack nuclear progesterone receptor (PGR) and represent a novel pharmaceutical target for new contraceptives. Little is known, however, regarding the expression and localization of PGRMC1 in the primate cervix. The goal of this study was to compare the expression of PGR and PGRMC1 in the rhesus macaque cervix during the proliferative and secretory phase of the menstrual cycle.

Abstract 465: Progestin-driven regulatory T cells directly promote an aggressive and metastatic phenotype in triple-negative breast cancer

Abstract The immune system plays key roles in the recognition and elimination of most tumors. Progesterone (Pg) can shape the immune response favoring a tolerogenic rather than a pro-inflammatory adaptive response. As hormone replacement therapies, supplementation and hormone-based contraceptives have been associated with an increase of malignant breast neoplasia we decided to investigate how progestins can regulate different key immune cell populations in the tumor microenvironment and their influence to tumor progression. To address this issue we used the highly metastatic, triple-negative 4T1 breast tumor. Balb/c mice treated with Pg or its synthetic analog, medroxyprogesterone acetate, showed an increased frequency of Foxp3+ regulatory T cells (Tregs) in draining lymph nodes (DLN) and an impaired antitumor response elicited by a decreased production of IL-17 and IFN-γ by CD4+ T cells (p&amp;lt;0,01). Tregs in DLN of progestin-treated animals showed increased expression of CD44 and CTLA-4. CD44+ Tregs were more suppressive in vitro than their CD44- counterpart. Concordantly, progestin-treated tumor-bearing mice showed a higher frequency of CD44+ Tregs and PD1+TIM3+ ‘exhausted’ CD8 T cells in the tumor (p&amp;lt;0,05). Both progestins favored in vitro Treg stable differentiation and expansion which resulted in a more robust suppression activity (p&amp;lt;0,01). Progestin-induced Treg differentiation could not be inhibited by the nuclear progesterone receptor (nPR) antagonist, Zk230211. Interestingly, T cells did not express detectable levels of nPR by qPCR but did express membrane progesterone receptor alfa, gamma and progesterone receptor membrane component type 1. In the presence of progestins, in vitro differentiated Tregs expressed higher levels of RANKL, a protein involved in breast stem cell maintenance and malignant transformation of tumors. When cocultured, ex vivo sorted- or in vitro differentiated Tregs increased 4T1 cells invasiveness both in vitro and in vivo following the downregulation of E-cadherin and increased expression of Snail (p = 0,05). Concordantly we observed a stable expansion of the CD44+ stem cell-like population when cocultured with progestin-iTregs. The CD44+ 4T1 population formed tumor spheres, exhibited an EMT molecular signature and had augmented tumorigenicity and a metastatic phenotype. Although primary tumor volume was not altered, the number of lung metastases was notably increased in animals treated with progestins (p&amp;lt;0,01). Additonally, overall survival of mice was decreased when injected with 4T1 cells cocultured with progestin-induced Tregs compared with 4T1 cocultured with control Tregs. Our findings highlight the relevance of progestins in modulating immunoregulatory checkpoints and harnessing immunosurveillance in the tumor microenvironment and suggest a mechanism through which Tregs could directly promote a metastatic and aggressive phenotype on tumor cells. Citation Format: Tomas Dalotto Moreno, Juan Pablo Cerliani, Diego Omar Croci, Santiago Patricio Mendez-Huergo, Florencia Moses, Gabriel Adrian Rabinovich, Mariana Salatino. Progestin-driven regulatory T cells directly promote an aggressive and metastatic phenotype in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2015-465

Abstract 2774: Androgen receptor is an independent prognostic marker of breast cancer in ethnically diverse women from Chicago

Abstract Androgen receptor (AR) has been demonstrated to be a prognostic marker in invasive breast cancer; higher expression levels of AR is associated with better prognosis among breast cancer patients. The goal of this analysis was to examine the extent to which AR was associated with specific breast cancer subtypes, and whether it could serve as an additional prognostic marker above and beyond breast cancer subtype, in an ethnically diverse sample of 287 patients (86 Non-Hispanic White, 84 Hispanic and 116 African American). Immunohistochemical analysis was performed on tissue microarrays constructed from invasive breast cancer samples obtained from the Breast Cancer Care in Chicago study. Tissue samples were tested for the expression of estrogen receptor (ER), progesterone receptor (PR), HER2. From these results, breast cancers were classified as Luminal A (ER+/PR+/HER2-), Luminal B (ER+/PR+/HER2+), HER2 enriched (ER-/PR-/HER2+), and triple negative (TN) (ER-/PR-/HER2-). Tumor tissues were also analyzed for the expression of other proliferative and apopototic markers (i.e. Ki67, P53 and bcl2) as well as BRCA1 protein expression. AR expression was evaluated based on the percentage of positive tumor cells and staining intensity using the H-score. The H score is a product of the percentage of cells (0-100%) in each intensity category (0, 1+, 2+ and 3+). The final score is on a continuous scale between 0 and 300. AR expression was then classified as low (&amp;lt;60%) or high (≥60%) using the mean of the AR score as a cutoff. Among the 287 breast cancer cases, 59% were expressing low levels of AR while 41% were expressing high AR. The percentage of tumor samples with high AR expression was much higher for luminal A (52%) than for luminal B, HER2, and TN subtypes (43, 29, and 9%, respectively(P&amp;lt;0.0001). High AR expression significantly correlated with early stage (P = 0.01), lower nuclear grade (P&amp;lt;0.0001), ER and PR positivity (P&amp;lt;0.0001), bcl2 positivity(P&amp;lt;0.0001), higher levels of BRCA1 expression (P&amp;lt;0.0001), lower Ki67 index (P&amp;lt;0.0001), and lower P53 expression (P&amp;lt;0.0001). A hormone receptor expression score (HRES) with a high internal reliability (Cronbach's alpha = 0.7035) was created based on standardized variables for ER, PR and AR expression. Using an ordinal logistic regression model, the HRES was a highly significant predictor of tumor grade (P&amp;lt;0.0001). In conclusion, in this multi-ethnic sample of breast cancer patients, we found that AR was a significant independent predictor of less aggressive, lower grade disease, even after accounting for molecular subtype. AR might serve as a useful additional marker for the classification of breast cancer into molecular subtypes. Citation Format: Abeer M. Mahmoud, Umaima Al-alem, Virgilia Macias, Jacob K. Kresovich, Andre Kajdacsy-Balla, Elizabeth L. Wiley, Garth H. Rauscher. Androgen receptor is an independent prognostic marker of breast cancer in ethnically diverse women from Chicago. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2774. doi:10.1158/1538-7445.AM2015-2774

Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo–produced and in vitro–produced embryos

Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, β, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, β, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology.

The Role of a Mitochondrial Progesterone Receptor (PR-M) in Progesterone Action.

Historically, progesterone functions to regulate gene expression via two nuclear progesterone receptors (PR-B and PR-A). Yet, actions of progesterone independent of gene regulation have been observed for decades. These are based on progesterone induced cellular events that occur too rapidly to involve gene transcription or in cells lacking active gene transcription such as mature spermatozoa. Understanding of these "nongenomic" effects has been slowed by the lack of identification of specific receptors. Previous discovery of a mitochondrial progesterone receptor, PR-M, has opened up the possibility of direct, ligand-dependent modulation of mitochondrial activity. In this article, we review the current knowledge of PR-M and speculate on possible physiologic and pathophysiologic actions.

Specific Role of Membrane Progesterone Receptors α and β on Ventilation during Sleep in Female Mice.

We recently showed that deletion of nuclear progesterone receptors disrupts breathing during sleep. Here we tested the hypotheses that deletion of membrane progesterone receptors (mPR) α and β in the medulla‐brain stem regions decreases ventilation and increases breathing instabilities during sleep. Female mice were chronically instrumented and small interfering RNA (siRNA) against mPRαor mPRβ (or a control solution) were infused in the fourth ventricle for 2 weeks. Ventilation (Ve) and CO2 production (VCO2) were continuously recorded (whole body plethysmography) during 4 h (9:00‐13:00) in normoxia. Respiratory frequency, tidal volume, minute ventilation and the frequency of spontaneous and post‐sigh apneas were determined during sleep, defined as periods of stable breathing pattern lasting at least 10 minutes. Compared to control, deletion of mPRα decreased Ve and Ve/VCO2 during sleep, but did not affect apnea or sigh frequency. The deletion of mPRβ had no effect on Ve but decreased VCO2, increased Ve/VCO2 and increased the frequency of spontaneous and post‐sigh apneas. Specific deletions of mPRα and mPRβ have been verified by immunohistochemistry on serial brainstem slices. Our results show that mPRα and β play specific role on respiratory control during sleep, and suggest that these receptors mediate some of the respiratory effects of progesterone. Founded by CIHR (MOP‐102715).

An estrogen-induced endometrial hyperplasia mouse model recapitulating human disease progression and genetic aberrations.

Endometrial hyperplasia (EH) is a condition originating from uterine endometrial glands undergoing disordered proliferation including the risk to progress to endometrial adenocarcinoma. In recent years, a steady increase in EH cases among younger women of reproductive age accentuates the demand of therapeutic alternatives, which emphasizes that an improved disease model for therapeutic agents evaluation is concurrently desired. Here, a new hormone-induced EH mouse model was developed using a subcutaneous estradiol (E2)-sustained releasing pellet, which elevates the serum E2 level in mice, closely mimicking the effect known as estrogen dominance with underlying, pathological E2 levels in patients. The onset and progression of EH generated within this model recapitulate a clinically relevant, pathological transformation, beginning with disordered proliferation developing to simple EH, advancing to atypical EH, and then progressing to precancerous stages, all following a chronologic manner. Although a general increase in nuclear progesterone receptor (PR) expression occurred after E2 expression, a total loss in PR was noted in some endometrial glands as disease advanced to simple EH. Furthermore, estrogen receptor (ER) expression in the nucleus of endometrial cells was reduced in disordered proliferation and increased when EH progressed to atypical EH and precancerous stages. This EH model also resembles other pathological patterns found in human disease such as leukocytic infiltration, genetic aberrations in β-catenin, and joint phosphatase and tensin homolog/paired box gene 2 (PTEN/PAX2) silencing. In summary, this new and comprehensively characterized EH model is cost-effective, easily reproducible, and may serve as a tool for preclinical testing of therapeutic agents and facilitate further investigation of EH.