Progesterone Receptor mRNA Expression Research Articles

Abstract 2791: Establishment of immortalized endometrial cell lines using Sendai virus

Abstract Objective: To establish endometrial immortalized cell lines using the Sendai virus and analyze their characteristics. Methods: The study included patients with benign ovarian tumors and endometrioid carcinoma who had regular menstrual cycles. Endometrial cells were collected and separated into epithelial and non-epithelial cells using flowcytometry (FCM) (CD326 (EpCAM) antibody). Furthermore, the isolated cells were infected with Sendai virus carrying three immortalization genes (Bmi-1, hTERT, and SV40T) to create immortalized cell lines. The cells were characterized as epithelial cells by immunocytochemistry (ICC), and receptor expression was assessed by reverse transcription PCR (RT-PCR) and ICC. Non-epithelial cells from the patients with endometrial carcinoma were evaluated using ICC. Results: The immortalized cell lines were capable of more than 20 passages. Immortalized endometrial epithelial cells tested positive for anti-Keratin antibodies and negative for anti-Vimentin antibodies by ICC. Additionally, FCM detected these cells as positive for anti-EpCAM antibody, confirming that they were epithelial cells. RT-PCR confirmed mRNA expression of estrogen α-receptor and progesterone receptor after 20 passages and ICC confirmed protein expression of them after 10 passages, establishing an endometrial epithelial immortalized cell line. Non-epithelial cells isolated from patients with endometrial carcinoma tested positive for anti-α-smooth muscle actin and anti-Vimentin antibodies by ICC, indicating their characterization as cancer-associated fibroblasts. Conclusion: We successfully established an immortalized cell line of endometrial epithelial cells using Sendai virus and generated cancer-associated fibroblasts. Citation Format: Kohei Hikino, Hiroaki Komatsu, Genki Hichiwa, Kanako Kazuki, Yasuhiro Kazuki, Hiroyuki Kugoh, Masayo Okawa, Yuki Ida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Shinya Sato, Fuminori Taniguchi. Establishment of immortalized endometrial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2791.

Endometrial mRNA expression of key inflammatory genes and progesterone receptor in uterine tissue of bitches with endometritis

Our study aimed to evaluate the gene transcription of Cyclooxygenase 1 (COX1), cytokines (Interleukin 6 [IL6], Interleukin 8 [IL8], and Leukemia Inhibitory Factor [LIF]), and Progesterone Receptor (PR) in clinically healthy bitches with a normal endometrium and with endometritis, without the presence of cystic endometrial hyperplasia. Forty-eight mixed breed bitches in dioestrus were used. Uterine biopsies were collected for diagnosis of a normal endometrium (n=15), and endometritis (n=30). Three samples were excluded from the study (two bitches with cystic endometrial hyperplasia and one bitch with atrophy). Samples were collected by cytobrush to quantify mRNA by qPCR. Data were analyzed using a generalized mixed model. COX1, IL6, and IL8 mRNA expression in bitches with endometritis was significantly higher than in a normal endometrium (P=0.0002, P=0.002, and P=0.003, respectively). In contrast, LIF and PR mRNA expression in bitches with endometritis was significantly lower than with a normal endometrium (P=0.006 and P=0.001, respectively). IL6 (AUC 0.67) was the most favorable biomolecular marker for predicting endometritis in bitches. There was an evident change in the mRNA expression of COX1, cytokines, and PR in clinically healthy bitches with a normal endometrium and endometritis without cystic endometrial hyperplasia. These results suggest that biomolecular markers such as COX1, IL6, IL8, LIF, and PR could help diagnose endometritis in a bitch.

Effects of mifepristone, a model compound with anti-progestogenic activity, on the development of African clawed frog (Xenopus laevis)

The objective of this study was to assess the effects of a model substance with anti-progestogenic activity on development of African clawed frog (Xenopus laevis) from tadpole to juvenile stage. Mifepristone, a synthetic progesterone receptor-blocking steroid hormone used in medicine as an abortifacient, was chosen as a model compound with anti-progestogenic activity. In the experiment, African clawed frog tadpoles were exposed to mifepristone at three concentrations (2, 21, and 215 ng L−1). A control group was exposed to dimethyl sulfoxide (DMSO; 0.001 %). The experiment started when tadpoles reached stages 47–48 according to Nieuwkoop and Faber (NF; 1994) and continued until stage NF 66, when metamorphosis was complete. Exposure to mifepristone had no significant effect on the rate of tadpole development, occurrence of morphological anomalies, weight, body length, or sex ratio. Mortality was within an acceptable range of 0–3.6 % throughout the test and did not differ among the groups. Histopathological examination of the gonads and thyroid gland revealed no significant changes. Therefore, we can conclude that mifepristone had no negative effect on development of the African clawed frog up to juvenile stage. Nevertheless, at the highest tested mifepristone concentration (215 ng L−1), gene expression analysis revealed up-regulation of mRNA expression of nuclear progesterone receptor (npr), membrane progesterone receptor (mpr), estrogen receptor beta (esrβ), and luteinizing hormone (lh) in the brain–pituitary complex of exposed frogs at stage NF 66. Higher mRNA expression of npr was also found in frogs exposed to 22 ng L−1 mifepristone compared to the solvent control. These findings confirmed the anti-progestogenic activity of mifepristone in frogs because the up-regulation of progesterone receptors occurs if progesterone availability in the body is reduced. All the observed changes in combination may have negative consequences for reproduction and reproductive behavior later in life.

Jiawei Shoutai Pill promotes decidualization by regulating the SGK1/ENaC pathway in recurrent spontaneous abortion

Ethnopharmacological relevanceJiawei Shoutai Pill (JWSTW) is a traditional herbal formula for recurrent spontaneous abortion (RSA). Although JWSTW significantly improves the clinical symptoms of RSA patients, its molecular mechanism remains unclear. Aim of studyThis study evaluated the expression and function of the serum/glucocorticoid regulated kinase 1/epithelial sodium channel (SGK1/ENaC) pathway and decidualization level in RSA patients and mice. It also investigated the therapeutic effects and potential mechanisms of JWSTW. Materials and methods30 early RSA patients and 30 normal pregnant women undergoing induced abortion during the same period were included in the study. Decidual tissues were collected, and HE staining, immunohistochemistry, Western blot, and RT-PCR were used to detect protein and mRNA expression levels of SGK1, ENaC-a, estrogen Rreceptor β (ERβ), and progesterone receptor (PR) in patients’ decidual tissues. Protein expression levels of prolactin receptor (PRLR) and insulin-like growth factor binding protein 1 (IGFBP-1) were also detected. A classical RSA mouse model was constructed, and the mice were randomly divided into four groups: normal, model, dydrogesterone (DQYT) (0.33 g/kg/d), and JWSTW (1.66 g/kg/d). The normal and model groups received the same volume of distilled water by gavage for 8 and 14 days after pregnancy. On the 14th day of pregnancy, the embryonic loss rate of each group, the number of offspring born to naturally delivered mice, and the protein or mRNA expression levels of key factors of the SGK1/ENaC pathway (SGK1, ENaC-a, ERβ, and PR), decidual proliferation marker (Ki67), mesenchymal-epithelial transition (E-cadherin and Vimentin), and decidualization markers (PRLR and IGFBP-1) in mouse decidual tissue on the eighth day of pregnancy were observed. ResultsThe decidual tissue structure of RSA patients was abnormal. Immunohistochemical analysis revealed significantly reduced positive expression of SGK1, ENaC-a, ERβ, and PR proteins in the decidual tissue of RSA patients (P < 0.001). Western blot and RT-PCR analyses demonstrated significantly decreased protein and mRNA expression of SGK1, ENaC-a, ERβ, and PR in the decidual tissue of RSA patients (all P < 0.05). Additionally, protein expression of PRLR and IGFBP-1 was significantly reduced (both P < 0.001). The RSA mouse model exhibited a significant increase in embryo loss rate and decreased litter size (both P < 0.001). Treatment with DQYT and JWSTW rescued the embryo loss rate and litter size to varying extents (all P < 0.05). The protein or mRNA expression levels of SGK1, ENaC-a, ERβ, PR, Ki67, E-cadherin, vimentin, PRLR, and IGFBP-1 in RSA mice were improved to different degrees after treatment with DQYT and JWSTW (all P < 0.05). ConclusionsAbnormal SGK1/ENaC signaling pathway regulation is closely associated with early endometrial damage in RSA patients. JWSTW promotes endometrial proliferation and mesenchymal-epithelial transition through the SGK1/ENaC signaling pathway, improving endometrial shedding. Consequently, JWSTW is a potential treatment for RSA.

Kelulut Honey Regulates Sex Steroid Receptors in a Polycystic Ovary Syndrome Rat Model

Reproductive and metabolic anomalies in polycystic ovary syndrome (PCOS) have been associated with the dysregulation of sex steroid receptors. Kelulut honey (KH) has been shown to be beneficial in PCOS-induced rats by regulating folliculogenesis and the oestrus cycle. However, no study has been conducted to evaluate KH's effect on sex steroid receptors in PCOS. Therefore, the current study examined the effects of KH, metformin, or clomiphene alone and in combination on the mRNA expression and protein distribution of androgen receptor (AR), oestrogen receptor α (ERα), oestrogen receptor β (ERβ), and progesterone receptor (PR) in PCOS-induced rats. The study used female Sprague-Dawley rats, which were treated orally with 1 mg/kg/day of letrozole for 21 days to develop PCOS. PCOS-induced rats were then divided and treated orally for 35 days with KH, metformin, clomiphene, KH + metformin, KH+ clomiphene and distilled water. In this study, we observed aberrant AR, ERα, ERβ and PR expression in PCOS-induced rats compared with the normal control rats. The effects of KH treatment were comparable with clomiphene and metformin in normalizing the expression of AR, ERα, and ERβ mRNA. However, KH, clomiphene and metformin did not affect PR mRNA expression and protein distribution. Hence, this study confirms the aberrant expression of sex steroid receptors in PCOS and demonstrates that KH treatment could normalise the sex steroid receptors profile. The findings provide a basis for future clinical trials to utilize KH as a regulator of sex steroid receptors in patients with PCOS.

Postnatal exposure to a glyphosate-based herbicide interferes with the development and growth of the mammary gland of pre-pubertal Ewe lambs

The aim of the present study was to evaluate whether early postnatal exposure to a glyphosate-based herbicide (GBH) alters pre-pubertal mammary development in Friesian lambs. To this end, from postnatal day 1–14, ewe lambs were exposed subcutaneously or orally to GBH (2 mg/kg bw/day) or vehicle (control) and mammary gland biopsies were obtained at 45 days of age. GBH-exposed lambs exhibited larger mammary ducts and less area occupied by terminal duct lobular units than controls, accompanied by an increase in the area of adipocytes in the mammary stroma. Lambs subcutaneously exposed to GBH showed increased protein expression of estrogen receptor alpha; however, both GBH-exposed groups had decreased mRNA expression of this receptor. Control lambs showed nuclear progesterone receptor (PR) protein expression, whereas GBH-exposed animals showed cytoplasmic PR expression; both GBH-exposed groups exhibited decreased mRNA expression of PR. GBH-exposed lambs also had decreased epithelial cell proliferation. Regarding insulin-like growth factors, both groups showed similar IGF-1 mRNA and protein expression but decreased expression of its receptor, and increased IGFBP5 expression. In addition, phosphorylated AKT was only observed in the mammary gland of control lambs. Our results show that early postnatal exposure to GBH, regardless of the exposure route, affects the IGF-1 system and the AKT/protein kinase B pathway, interfering with steroid hormone receptor expression and cell proliferation. This consequently modifies the growth and development of the pre-pubertal mammary gland of Frisian lambs.

OR16-2 Steroid Receptor Co-Activator Complexes Regulate Metabolic PFKFB Enzymes to Drive Therapy Resistant ER+ Breast Cancer

Abstract Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the survival and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in recurrent tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. We previously showed that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. PELP1 interacts with PFKFB proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB inhibitors in combination with ER targeted therapies blocked proliferation and tumorsphere formation in multiple models of advanced breast cancer, including paclitaxel (TaxR) and tamoxifen (TamR) resistant models and ER+ patient-derived organoids (PDxO). Chemotherapy and endocrine therapy resistant models contained increased levels of cytoplasmic PELP1, PELP1/SRC-3 complexes, and phenocopied PELP1/SRC-3/PFKFB biology and CSC behavior. To better understand CSC-mediated pathways in the context of therapy resistance, we performed RNA-seq on 3D-cultured TaxR tumorspheres to enrich for CSCs. Pathway analysis revealed that TaxR tumorspheres upregulate stem (Sox2-Oct4-Nanog), HIF, and progesterone mediated pathways. Progesterone receptor (PR) is a known contributor to CSC populations, and we observed increased PR mRNA and protein expression in TaxR cells grown as tumorspheres. Additionally, in vivo emergence of circulating tumor cell (CTC) populations was observed in ER+ TaxR mammary intraductal (MIND) xenografts. Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Our findings are applicable to therapy resistant models, indicating that SR co-activator complexes are a key mediator of resistance in ER+ breast cancer and may cooperate with PR to promote therapy resistant CSCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer. Presentation: Sunday, June 12, 2022 11:15 a.m. - 11:30 a.m.

Zearalenone Promotes Uterine Development of Weaned Gilts by Interfering with Serum Hormones and Up-Regulating Expression of Estrogen and Progesterone Receptors.

In this study, we aimed to assess the effect of diet ZEA on serum hormones, the location and expression of estrogen receptor ERα/β and progesterone receptor (PR) of the uterus in weaned piglets and to reveal the mechanism underneath. A total of 40 healthy weaned gilts were randomly allocated to basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0) and 1.5 (ZEA1.5) mg ZEA/kg and fed individually for 35 days. Meanwhile, the porcine endometrial epithelial cells (PECs) were incubated for 24 h with ZEA at 0 (Control), 5 (ZEA5), 20 (ZEA20) and 80 (ZEA80) μmol/L, respectively. The results showed that nutrient apparent digestibility (CP and GE), nutrient apparent availability (ME/GE, BV and NPU), the uterine immunoreactive integrated optic density (IOD), relative mRNA and protein expression of ER-α, ER-β and PR and the relative mRNA and protein expression of ER-α and ER-β in PECs all increased linearly (p < 0.05) with ZEA. Collectively, ZEA can interfere with the secretion of some reproductive hormones in the serum and promote the expression of estrogen/progesterone receptors in the uterus and PECs. All these indicate that ZEA may promote the development of the uterus in weaned gilts through estrogen receptor pathway.

The effect of pre-analytical variables on downstream application and data analysis of human endometrial biopsies.

STUDY QUESTIONWhat are the effects of pre-analytical variables on the downstream analysis of patient-derived endometrial biopsies?SUMMARY ANSWERThere are distinct differences in the protein levels of the master regulator of oxygen homeostasis, hypoxia-inducible factor-1-alpha (HIF1α), and the protein and mRNA levels of three related genes, carbonic anhydrase 9 (CA9), vascular endothelial growth factor A (VEGFA) and progesterone receptor (PR) in human endometrial biopsies, depending on the pre-analytical variables: disease status (cancer vs benign), timing of biopsy (pre- vs post-hysterectomy) and type of biopsy (pipelle vs full-thickness).WHAT IS KNOWN ALREADYPatient-derived biopsies are vital to endometrial research, but pre-analytical variables relating to their collection may affect downstream analysis, as is evident in other tissues.STUDY DESIGN, SIZE, DURATIONA prospective observational study including patients undergoing hysterectomy for endometrial cancer (EC) or benign indications was conducted at a large tertiary gynaecological unit in the UK. Endometrial biopsies were obtained at different time points (pre- or post-hysterectomy) using either a pipelle endometrial sampler or as a full-thickness wedge biopsy.PARTICIPANTS/MATERIALS, SETTING, METHODSThe changes in HIF1α, CA9, VEGFA and PR protein levels were measured by semi-quantitative analysis of immunostaining, and the expression levels of three genes (CA9, VEGFA and PR) were investigated by quantitative real-time PCR, in endometrial biopsies from 43 patients undergoing hysterectomy for EC (n = 22) or benign gynaecological indications (n = 21).MAIN RESULTS AND THE ROLE OF CHANCEAn increase in HIF1α immunostaining was observed in EC versus benign endometrium (functionalis glands) obtained pre-hysterectomy (P < 0.001). An increase in CA9 immunostaining was observed in EC versus benign endometrial functionalis glands at both pre- and post-hysterectomy time points (P = 0.03 and P = 0.003, respectively). Compared with benign endometrial pipelle samples, EC samples demonstrated increased mRNA expression of CA9 (pre-hysterectomy P < 0.001, post-hysterectomy P = 0.008) and VEGFA (pre-hysterectomy P = 0.004, post-hysterectomy P = 0.002). In benign uteri, HIF1α immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.009), VEGFA immunoscores (functionalis glands, P = 0.03 and stroma, P = 0.01) and VEGFA mRNA levels (P = 0.008) were increased in matched post-hysterectomy versus pre-hysterectomy samples. Similarly, in EC, an increase in VEGFA immunoscores (epithelial and stromal) and VEGFA mRNA expression was observed in the matched post-hysterectomy versus pre-hysterectomy biopsies (P = 0.008, P = 0.004 and P = 0.018, respectively). Full-thickness benign post-hysterectomy endometrial biopsies displayed increased VEGFA (P = 0.011) and PR (P = 0.006) mRNA expression compared with time-matched pipelle biopsies.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis descriptive study explores the effect of pre-analytical variables on the expression of four proteins and three hypoxia-related genes in a limited number of endometrial biopsies from patients with EC and benign controls. Due to the small number, it was not possible to investigate other potential variables such as menstrual cycle phase, region-specific differences within the endometrium, grade and stage of cancer, and surgical technicalities.WIDER IMPLICATIONS OF THE FINDINGSCareful consideration of the effects of these pre-analytical variables is essential when interpreting data relating to human endometrial biopsies. A standardized approach to endometrial tissue collection is essential to ensure accurate and clinically transferrable data.STUDY FUNDING/COMPETING INTEREST(S)The authors have no conflicts of interest to declare. The work included in this manuscript was funded by Wellbeing of Women project grants RG1073 and RG2137 (D.K.H.), Wellbeing of Women Entry-Level Scholarship ELS706 and Medical Research Council MR/V007238/1 (A.M./D.K.H.), Liverpool Women’s Hospital Cancer Charity (M.A.) and University of Liverpool (L.B., L.R. and E.N.).

Establishment and comparison of two methods to produce a rat model of mammary gland hyperplasia with hyperprolactinemia

This study aimed to establish and compare models of mammary gland hyperplasia (MGH) with hyperprolactinemia (HPRL) using two different methods. The models provide information on the relationship between mammary gland hyperplasia and associated hormones. Model A was constructed using intramuscular injections of estradiol benzoate injection (EBI), followed by progesterone (P), and then metoclopramide dihydrochloride (MDI). Model B was designed by administering MDI, follow by EBI, and then P intramuscularly. Model B showed higher MGH progression compared with model A. Notably, increase in estradiol (E2) was negatively correlated with prolactin (PRL) secretion. However, PRL levels in model B were significantly higher compared with the levels in model A. Estrogen (ER), prolactin receptor (PRLR), and progesterone receptor (PR) mRNA and protein expression levels in model B rats were positively correlated with changes in the corresponding hormone levels. However, E2, P, and PRL levels in model A showed no direct relationship with levels of the mRNAs of related hormones and protein expression levels. Our results suggest that model B is an appropriate model of MGH with HPRL that can be used to perform further studies about the interactions of the E2, P, and PRL hormones in this disorder.

Progesterone Receptor Together with PKCα Expression as Prognostic Factors for Astrocytomas Malignancy.

IntroductionAstrocytomas are the most common and aggressive primary brain tumors, and they are classified according to the degree of malignancy on a scale of I to IV, in which grade I is the least malignant and grade IV the highest. Many factors are related to astrocytomas progression as progesterone receptor (PR), whose transcriptional activity could be regulated by phosphorylation by protein kinase C alpha (PKCα) at the residue Ser400. Our aim was to investigate if PR phosphorylation together with PKCα expression could be used as a prognostic factor for astrocytomas malignancy.MethodsBy immunofluorescence, we detected the content of PKCα, PR and its phosphorylation at Ser400 in 46 biopsies from Mexican patients with different astrocytoma malignancy grades; by bioinformatic tools using TCGA data, we evaluated the expression of PR and PKCα mRNA according to astrocytoma malignancy grades. For all statistical analyses, significance was p<0.05.ResultsWe detected a positive correlation between the tumor grade and the content of PKCα, PR and its phosphorylation at Ser400, as well as the intracellular colocalization of these proteins. Interestingly, using an in silico assay, we found that the PR and PKCα expression at mRNA level has an inverse ratio with astrocytomas tumor grade.DiscussionThese results indicate that PR and its phosphorylation at Ser400 site, as well as PKCα and their colocalization, could be considered as possible malignancy biomarkers for astrocytomas grades I–IV.

Hydrolyzed seawater pearl tablet modulates the immunity via attenuating Th1/Th2 imbalance in an immunosuppressed mouse model.

To investigate whether Hydrolyzed Seawater Pearl tablet (HSPT) could modulate the Th1/Th2 imbalance in an immunosuppressed mouse model with Th1 to Th2 shift induced by Cyclosporine A (CsA) which can be used in the clinical treatment of Th2 to Th1 shift diseases, and explore the possible mechanism for the adjuvant therapeutic efficacy of HSPT on recurrent respiratory infections (RRI) and acquired immune deficiency syndrome (AIDS). The mice were randomly divided into six groups of five animals each, namely normal group, model group, lentinan polysaccharide tablet (LPT) group and three HPST treated groups. HPST treated groups were administered with HPST (0.51, 1.02, 2.04 g/kg) via intragastric gavage (i.g) for 30 consecutive days. LPT used as reference drug for positive control, LPT group was administered with LPT (8.2 mg/kg) for 30 consecutive days. Normal group and model group were received distilled water. The animals in model group, LPT group and HPST treated groups were injected intraperitoneally with CsA (50 mg/kg) to establish the immunosuppressed mice model with Th1 to Th2 shift on the 20th, 22nd and 24th day, one hour after the administration of the respective treatment. Animals were sacrificed one hour after the last administration to collect blood and splenic tissue. The proportion of T cells including CD8+ and CD4+ T cells, Th1 and Th2 in peripheral blood of experimental mice were measured by flow cytometric. The protein level in serum and mRNA level in splenic tissue of experimental mice for interleukin (IL)-2, IL-12, interferon-γ (IFN-γ), IL-4, IL-6, IL-10 and IL-13 were measured by enzyme linked immunosorbent assay and fluorescence quantitative polymerase chain reaction respectively. HSPT elevated the proportion of T cells including both CD8+ and CD4+ T cells, in which the proportion of Th1 and Th2 cells increased, while the ratio of Th1/Th2 cells decreased in peripheral blood of the immunosuppressed mouse model with Th1 to Th2 shift induced by CsA. Furthermore, HSPT elevated both protein and mRNA level of Th1-type cytokines IL-2 and IFN-γ, while had no significant effect on protein and mRNA level of Th1-type cytokine IL-12 and Th2-type cytokines IL-4, IL-6, IL-10, IL- 13 in mouse model. Our findings suggest that HSPT can increase proportion of T cells including both CD8+ and CD4+ T cells and induce Th2 to Th1 shift in both cells and cytokines, which probably was the mechanism to account for the adjuvant therapeutic efficacy of HSPT on RRI and AIDS.

226-LB: Elimination of the Progesterone Receptor in ß Cells Improves ß-Cell Function and Glucose Tolerance during Pregnancy and Diet-Induced Insulin Resistance in Female Mice

We showed that the gonadal hormones estradiol and testosterone influence islet β-cell function and survival in a sex-specific manner via estrogen and androgen receptors expressed in islet β-cells. The progesterone receptor (PR) is a ligand-activated transcription factor and is believed to be involved in the β-cell adaptation to pregnancy. Leveraging public single-cell RNA-seq datasets we observe PR mRNA expression in α- β- and γ-cells in male and female human pancreatic endocrine cells. Using immunohistochemistry, we observe that, unlike in the uterus where PR is nuclear, PR is localized in the cytoplasm of β-cells in male and female human and mouse islets. We investigated the role of progesterone and PR in β-cell physiology in male and female mice with conditional deletion of PR in β-cells (βPRKO) by crossing PR-Lox/lox and MIP-Cre transgenic mice. Male βPRKO mice exhibited no alteration in glucose homeostasis on normal chow (NC) or following western-diet (WD)-induced insulin resistance and obesity compared to littermate PR-Lox/lox controls. Similarly, NC-fed female βPRKO mice exhibited no abnormality of glucose or insulin homeostasis compared to littermate PR-Lox/lox controls. However, during pregnancy or WD feeding, female βPRKO mice developed enhanced IP-glucose-stimulated insulin secretion, associated with improved IP glucose tolerance compared to littermate PR-Lox/lox controls. These results reveal that PR is a cytosolic receptor in β-cells, and suggest that in conditions of high insulin demand, such as pregnancy and diet-induced insulin resistance, elimination of PR action in β-cells of females improves the ability of β-cells to produce insulin and lowers blood glucose. PR may be involved in the pathogenesis of gestational diabetes. Disclosure P. Mota de sa: None. M. Qadir: None. F. Mauvais-jarvis: None. Funding National Institutes of Health (DK074970, DK107444); U.S. Department of Veterans Affairs (BX003725)

Chlamydia inhibits progesterone receptor mRNA expression in SHT-290 cells.

SummaryChlamydia trachomatis is the most commonly diagnosed sexually transmitted infection in the UK, with over 200,000 positive diagnoses annually. The infection is thought to cause reproductive complications including problems in conceiving a pregnancy through to miscarriage and early or stillbirth. One potential reason Chlamydia may impact upon pregnancy is through disrupting the embryo implantation at the earliest stages of pregnancy is by altering the ability of specific cells that line the uterus called stromal cells to respond to the hormone progesterone, the hormone responsible for preparing the uterus for pregnancy. The results of this study showed that Chlamydial infection of these uterus lining stromal cells decreased the levels of specific progesterone sensitive markers which are associated with early embryo implantation, suggesting a loss of responsiveness to progesterone treatment. These changes were accompanied by a decrease in the levels of RNA for the progesterone receptor which is responsible for progesterone activity, suggesting that this is a potential mechanism through which Chlamydia could directly inhibit the effects of progesterone on uterine cells.

Discovery of novel 2H-chromene-3-carbonyl derivatives as selective estrogen receptor degraders (SERDs): Design, synthesis and biological evaluation

Selective estrogen receptor degraders (SERDs) not only block ERα activity but degrade this receptor at the same time and are effective in relapsed ERα positive breast cancer patients who have accepted other endocrine therapies. Herein, through scaffold hopping of coumarin skeleton, a series of 2H-chromene-3-carbonyl-based SERDs with phenyl acrylic acid group as the side chain were designed and synthesized. Compound XH04 containing 7-hydroxy-2H-chromene-3-carbonyl skeleton exhibited the most potent activities in 2D (IC50 = 0.8 μM) and 3D cells culture models (MCF-7) and had the best ERα binding affinity as well. Furthermore, the significant antiestrogen property of compound XH04 was confirmed by inhibiting the expression of progesterone receptor (PgR) mRNA in MCF-7 cells. On the other hand, the outgoing ERα degradation property of compound XH04 was qualitatively and quantificationally verified by immunofluorescence analysis and Western blot assay in MCF-7 cells. Besides, compound XH04 repressed the expression level of Ki67 in MCF-7 cells and induced the apoptosis increase of this tumor cells in a dose-dependent manner like approved-SERD fulvestrant (2), while compound XH04 exhibited better preliminary pharmacokinetics in human and rat liver microsomes in vitro and a lower LogD7.4 value than fulvestrant. And further molecular docking study revealed that compound XH04 possessed a proverbial and typical binding model with ERα like other reported SERD. All these results confirmed that 7-hydroxy-2H-chromene-3-carbonyl structure could be a feasible skeleton for design of ERα antagonists including SERDs and compound XH04 is a promising candidate for further development of ERα + breast cancer therapy agents.

Evaluation of Expression and Phosphorylation of Progesterone Receptor in Endometrial Stromal Cells of Patients with Recurrent Implantation Failure Compared to Healthy Fertile Women

Recurrent implantation failure (RIF) is the repeated failure of good-quality embryos in implantation process following several assisted reproduction cycles. Disruption of the endometrial receptivity is one of the main causes of RIF. Progesterone plays a pivotal role in the endometrial receptivity through the regulation of gene expression pattern by binding to its receptors in the endometrial cells. The aim of this study was to evaluate the expression level of progesterone receptor (PR) and its phosphorylated form in the endometrial stromal cells (eSC) of RIF patients and compare it to the eSC of healthy fertile women as control group. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, expression levels of PR mRNA, PR protein, and phospho-Ser294 PR protein were evaluated by quantitative real-time PCR and immunofluorescence staining, respectively. The results demonstrated a significant reduction in PR mRNA expression (P < 0.01.) and phospho-Ser294 PR protein (P < 0.05) level in RIF patients compared to the control group. These data for the first time suggest that the expression of PR and its phosphorylated form are impaired in RIF patients. Therefore, designing therapeutic methods for improving PR expression status and its regulation in the endometrium of RIF patients may help in improving the final reproductive outcomes of these cases.

Endometrial expression of key genes related to fertility in repeat breeder and non-repeat breeder cows.

The aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin-endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non-RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at -20°C until RT-qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non-RBC (3 and 25-fold change, p<.01 and p<.01, respectively), while the mRNA expression of PTGS2 was lower (1.56-fold change, p<.01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p<.05) and a negative correlation between PGR and PTGS2 (-0.49, p<.05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non-RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.

Effect of progesterone antagonist RU486 on uterine progesterone receptor mRNA expression,embryonic development and ovarian function during early pregnancy in pigs

Effect of progesterone antagonist RU486 on uterine progesterone receptor mRNA expression,embryonic development and ovarian function during early pregnancy in pigs

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10−9 M or 1 × 10−6 M BPA; or c) 1 × 10−12 M, 1 × 10−9 M or 1 × 10−6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. Total PR expression was also increased with 1 × 10−6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10−9 M BPA and 1 × 10−12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10−12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10−9 M BPA or 1 × 10−12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10−9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3β-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3β-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3ʹ-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.