P4 Receptor Research Articles

Changes in expression of ISG15, progesterone receptor and progesterone-induced blocking factor in ovine thymus during early pregnancy

Interferon-tau (IFNT) is the main signal for the maternal recognition of pregnancy in ruminants, and exerts its effects by stimulating the expression of interferon-stimulated genes, including the expression of interferon-stimulated gene15 kDa protein (ISG15). Progesterone (P4) exerts significant immune effects on the uterus during early pregnancy in ruminants that are partly mediated by progesterone-induced blocking factor (PIBF). The thymus is necessary for the normal development of immunologic function. In this study, thymuses were obtained on day 16 of the estrous cycle and on days 13, 16 and 25 of pregnancy (n = 6 for each group) from ewes. Our results showed that the expression of ISG15, P4 receptor (PGR) and PIBF mRNA and the expression of ISG15 and ISG15-conjugated proteins were upregulated in the thymuses during early pregnancy, and the 89-kDa PGR isoform and the 80-kDa PIBF variant were expressed constantly in the thymuses. However, there was no expression of the 60-kDa PGR isoform and the 62-kDa PIBF variant on day 16 of the estrous cycle. ISG15 and ISG15-conjugated proteins were limited to the epithelial reticular cells, capillaries and thymic corpuscles. This paper reports for the first time that early pregnancy exerts its effects on the thymus through IFNT and P4 in sheep.

Progesterone and cAMP synergize to inhibit responsiveness of myometrial cells to pro-inflammatory/pro-labor stimuli.

Progesterone (P4) acting through the P4 receptor (PR) isoforms, PR-A and PR-B, promotes uterine quiescence for most of pregnancy, in part, by inhibiting the response of myometrial cells to pro-labor inflammatory stimuli. This anti-inflammatory effect is inhibited by phosphorylation of PR-A at serine-344 and -345 (pSer344/345-PRA). Activation of the cyclic adenosine monophosphate (cAMP) signaling pathway also promotes uterine quiescence and myometrial relaxation. This study examined the cross-talk between P4/PR and cAMP signaling to exert anti-inflammatory actions and control pSer344/345-PRA generation in myometrial cells. In the hTERT-HMA/B immortalized human myometrial cell line P4 inhibited responsiveness to interleukin (IL)-1β and forskolin (increases cAMP) and 8-Br-cAMP increased this effect in a concentration-dependent and synergistic manner that was mediated by activation of protein kinase A (PKA). Forskolin also inhibited the generation of pSer344/345-PRA and expression of key contraction-associated genes. Generation of pSer344/345-PRA was catalyzed by stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Forskolin inhibited pSer344/345-PRA generation, in part, by increasing the expression of dual specificity protein phosphatase 1 (DUSP1), a phosphatase that inactivates mitogen-activated protein kinases (MAPKs) including SAPK/JNK. P4/PR and forskolin increased DUSP1 expression. The data suggest that P4/PR promotes uterine quiescence via cross-talk and synergy with cAMP/PKA signaling in myometrial cells that involves DUSP1-mediated inhibition of SAPK/JNK activation.

Abstract B37: Prevention of epithelial ovarian cancer by eliciting P4/RIPK1-dependent necroptosis

Abstract High-grade serous epithelial ovarian cancer (HGSOC) is the deadliest gynecologic disease, commonly diagnosed in peri/postmenopausal women. Annually, HGSOC accounts for over 140,000 deaths worldwide. Because we lack effective early detection methods, over 75% of HGSOC are diagnosed at stages III/IV, with abysmal 5-year survival rates of 37/25%, respectively. Recently, progesterone (P4) was observed to promote the elimination of p53-null cells from the fallopian tube by inducing necroptosis, a regulatable form of non-apoptotic cell death. Activation of necroptosis depends on activation of receptor-interacting kinase proteins 1 and 3 (RIPK1 and RIPK3). Several additional factors suggest that P4 may have a key protective role versus HGSOC; protection versus tumorigenesis is reduced at the time of menopause due to decreasing P4 levels. Several studies examining the use of a synthetic progesterone (medroxyprogesterone acetate, MPA) observed decreases in the incidence of HGSOC. Also, MPA had a significant clinical benefit in 20-40% of pretreated ovarian cancer patients. Further, 75% of HGSOC have decreased expression of progesterone receptor (PGR). With lower P4 at menopause, p53 mutated fallopian tube epithelial cells may therefore escape necroptosis and progress to HGSOC. All of the above information led to our hypothesis that activating P4 signaling will induce necroptosis in HGSOC tumor cells. Examining The Cancer Genome Atlas (TCGA) database for regulators of necroptosis, we found that increased expression of the P4 receptor (PGR), necroptosis effectors TNFα, and RIPK1/3 corresponded to significantly higher overall survival in 484 HGSOC cases. Using OVSAHO (BRCA2-deleted) cells, we show that P4 and/or TNFα treatment increases TNFα, RIPK1, and RIPK3 expression. In OVSAHO cells, we observed a significant decrease in cell number following treatment with P4 and/or TNFα. Also, we confirmed that P4 treatment was able to induce a necroptotic response distinct from classical apoptosis. Upon examination of normal fallopian tube tissue, borderline tumors, and advanced HGSOC, we detected expression of necroptosis effectors RIPK1 and 3, suggesting an intact necroptosis pathway. These observations point towards an HGSOC prevention strategy that favors the elimination of “founder” cells by activating necroptosis downstream of P4. These data may be particularly useful for the management of BRCA1/2 mutant patients, as these women experience menopause significantly earlier than noncarriers. Citation Format: Benjamin Bitler, Tomomi Yamamoto, Elise Bales, Zachary Watson, Kian Behbakht, Joshua Johnson. Prevention of epithelial ovarian cancer by eliciting P4/RIPK1-dependent necroptosis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B37.

Developmental profiles of progesterone receptor transcripts and molecular responses to gestagen exposure during Silurana tropicalis early development

Environmental gestagens are an emerging class of contaminants that have been recently measured in surface water and can interfere with reproduction in aquatic vertebrates. Gestagens include endogenous progestogens, such as progesterone (P4), which bind P4-receptors and have critically important roles in vertebrate physiology and reproduction. Gestagens also include synthetic progestins, which are components of human and veterinary drugs, such as melengestrol acetate (MGA). Endogenous progestogens are essential in the regulation of reproduction in mammalian species, but the role of P4 in amphibian larval development remains unclear. This project aims to understand the roles and the regulatory mechanisms of P4 in amphibians and to assess the consequences of exposures to environmental gestagens on the P4-receptor signaling pathways in frogs. Here, we established the developmental profiles of the P4 receptors: the intracellular progesterone receptor (ipgr), the membrane progesterone receptor β (mpgrβ), and the progesterone receptor membrane component 1 (pgrmc1) in Western clawed frog (Silurana tropicalis) embryos using real-time qPCR. P4-receptor mRNAs were detected throughout embryogenesis. Transcripts for ipgr and pgrmc1 were detected in embryos at Nieuwkoop and Faber (NF) stage 2 and 7, indicative of maternal transfer of mRNA. We also assessed the effects of P4 and MGA exposure in embryonic and early larval development. Endocrine responses were evaluated through transcript analysis of a suite of gene targets of interest, including: ipgr, mpgrβ, pgrmc1, androgen receptor (ar), estrogen receptor α (erα), follicle stimulating hormone β (fshβ), prolactin (prl), and the steroid 5-alpha reductase family (srd5α1, 2, and 3). Acute exposure (NF 12–46) to P4 caused a 2- to 5-fold change increase of ipgr, mpgrβ, pgrmc1, and ar mRNA levels at the environmentally relevant concentration of 195 ng/L P4. Acute exposure to MGA induced a 56% decrease of srd5α3 at 1140 ng/L MGA. We conclude that environmental exposure to P4 induced multiple endocrine-related transcript responses in amphibians; however, the differential responses of MGA suggest that the effects of MGA are not mediated through the classical P4 signaling pathway in S. tropicalis.

Progesterone as a Postnatal Prophylactic Agent for Encephalopathy Caused by Prenatal Hypoxic Ischemic Insult.

Brain damage caused by hypoxic ischemic insult during the perinatal period causes hypoxic ischemic encephalopathies (HIEs). Therapeutic hypothermia is indicated for HIE, but because the therapeutic burden is large for its limited therapeutic effectiveness, another strategy is needed. Progesterone (P4) plays a neuroprotective role through the actions of its metabolite, allopregnanolone (Allo), on P4 receptor, γ-aminobutyric acid type A receptors or both. We examined the therapeutic potential of P4 using a newborn rat model of HIE. Fetal rats were exposed to transient ischemic hypoxia by 30-minute bilateral uterine artery clamping on gestational day 18. After spontaneous birth, newborn pups were subcutaneously injected with P4 (0.10 or 0.01 mg), medroxyprogesterone acetate (MPA; 0.12 mg), or Allo (0.10 mg) through postnatal days (PDs) 1 to 9. Brain damage in the rats was assessed using the rotarod test at PD50. The HIE insult reduced the rats’ ability in the rotarod task, which was completely reversed by P4 and Allo, but not by MPA. Histological examination revealed that the HIE insult decreased neuronal (the cortex and the hippocampal CA1 region) and oligodendroglial cell density (the corpus callosum) through PD0 to PD50. The axon fiber density and myelin sheath thickness in the corpus callosum were also reduced at PD50. The time-course study revealed that P4 restored oligodendroglial cells by PD5, which was followed by neuroprotective action of P4 that lasted long over the injection period. These results suggest that P4 protects the neonatal brain from HIE insult via restoration of oligodendroglial cells.

Immunohistochemical localization of 3β-Hydroxysteroid dehydrogenase and progesterone receptors in the ovary and placenta during gestation of the placentotrophic lizard Mabuya sp (Squamata: Scincidae)

In squamates, progesterone (P) plays a key role in the inhibition of uterine mobility during egg retention in oviparous species, and during gestation in viviparous species. The corpus luteum (CL) is the main organ responsible for the production of P; however, in some species, the CL degenerates early and the P needed for gestation maintenance should be produced in other tissues. Mabuya sp (Scincidae) is a viviparous lizard with a prolonged gestation, it produces microlecithal eggs and, consequently, has an obligate placentotrophy related with a highly complex placenta. Its CL degenerates at early stages of gestation and therefore, other sources of P should exist. The aim of this study was to determine and localize by immunohistochemistry the production of P by detection of the enzyme 3β-Hydroxysteroid dehydrogenase (3β-HSD) and P receptors (PR) during gestation in the ovary and placenta of Mabuya sp. Positive and negative control sections were used. The ovary of this species localizes 3β-HSD and PR in the same tissues. The CL of the ovaries of females at early stages of gestation were positive for both molecules, whereas they did not localize from mid gestation to the end of pregnancy. Previtellogenic and vitellogenic follicles labelled for both molecules in the follicular epithelium and thecae. The placenta of Mabuya sp. demonstrated the potential for P production from mid gestation to the end of gestation in the uterine and chorionic tissues. PR were located in the uterine tissues throughout gestation, with a decrease towards its completion. Western blot analysis confirmed the presence of 3β-HSD mainly in the ovary of early pregnant females and in the placental tissues at mid gestation stages. Therefore, the chorioallantoic placenta of Mabuya sp. has an endocrine function producing the P needed for gestation and replacing the CL from mid gestation to the end of pregnancy.

Expression of membrane progestin receptors (mPRs) in the bovine corpus luteum during the estrous cycle and first trimester of pregnancy

Progesterone (P4) affects luteal cell function through nuclear P4 receptors and via nongenomic mechanisms, presumably involving membrane P4 receptors. There are 2 types of these receptors: progesterone receptor membrane component (PGRMC) and membrane progestin receptor (mPR), including mPR alpha (mPRα), beta (mPRβ), and gamma (mPRγ), which belong to the progestin and adipoQ receptor family (PAQR 7, 8, and 5, respectively). The aim of this study was to evaluate mRNA expression, protein expression, and localization of mPRα, mPRβ, and mPRγ in the bovine corpus luteum (CL) on days 2–5, 6–10, 11–16, and 17–20 of the estrous cycle as well as on weeks 3–5, 6–8, and 9–12 of pregnancy (n = 5/each period). The highest mPRα mRNA expression was found on days 11–16 (P < 0.05) and 17–20 (P < 0.001) of the estrous cycle compared with other stages of the estrous cycle and pregnancy. The mPRβ mRNA level was highest (P < 0.01) on days 11–20 of the estrous cycle and in all stages of pregnancy. mPRγ mRNA expression was highest (P < 0.001) on days 17–20 of the estrous cycle and also during weeks 9–12 of pregnancy compared with the other stages of the estrous cycle and pregnancy. Only the mPRα protein was changed during the estrous cycle; there were no significant differences in protein expression of mPRβ and mPRγ during the estrous cycle and pregnancy. Immunostaining for the mPRα, mPRβ, and mPRγ proteins was detectable in the CL sections at all stages of the estrous cycle and pregnancy. Strong positive staining was observed in small luteal cells; this reaction was less evident in large luteal cells. All proteins were also localized in endothelial cells of blood vessels. The obtained data indicate variable expression of mPRα, mPRβ, and mPRγ in bovine CL during the estrous cycle and first trimester of pregnancy and suggest that P4 may be involved in the regulation of CL function via these membrane receptors during both the estrous cycle and pregnancy.

Expression of progesterone receptor and progesterone-induced blocking factor in the spleen during early pregnancy in ewes

The spleen plays a key role in immune regulation. Progesterone (P4) exhibits different immunological effects through binding to different types of receptor. In the present study, splenic samples were obtained at day 16 of non-pregnancy and days 13, 16, and 25 of pregnancy to study the expression of P4 receptor (PGR) and P4-induced blocking factor (PIBF) in ewes through quantitative real-time PCR, Western blot and immunohistochemistry analysis. Our results showed that the relative expression levels of PGR and PIBF mRNA increased from day 13–16 in pregnant ewes, but decreased from day 16–25, and the 26kDa isoform of PGR and the 22kDa variant of PIBF were down-regulated in the spleens at day 25 of pregnancy compared with expression at day 16 of the oestrous cycle and days 13 and 16 of pregnancy. The immunohistochemistry test results confirmed that PGR and PIBF proteins were localized in the cytoplasm of cells in the capsule, trabeculae and splenic cords. This paper reports for the first time that the 26kDa isoform of PGR and the 22kDa variant of PIBF were down-regulated during early pregnancy, which may be essential for normal pregnancy in ewes.

161 Cumulus Cell Luteinization is Enhanced During Aging of Bovine Oocytes Matured In Vitro

Maturing mammalian oocytes can prevent luteinization of cumulus cells (CC) both in vivo and in vitro by regulating CC steroidogenesis (Gilchrist et al. 2004 Anim. Reprod. Sci. 82-83, 431-446; Ramirez et al. 2016 Anim. Reprod. 14, 280). However, when the oocyte attains the metaphase II stage, aging processes are accelerated and may impair protective abilities of CC. The aim of the present research was to study the luteinization of CC surrounding aging bovine oocytes and its susceptibility to prolactin (PRL), which performs a luteotropic function in mammals. We analysed (1) the steroidogenic activity of CC during the prolonged culture of cumulus–oocyte complexes (COC), and (2) the expression of genes related to luteinization in CC. Bovine COC were matured in TCM-199 containing 10% fetal calf serum (FCS), 10 μg mL−1 porcine FSH, and 10 μg mL−1 ovine LH. After 22 h of IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% FCS and cultured for 0, 12, or 24 h in the absence (Control) or presence of 50 ng mL−1 bovine PRL. Progesterone (P4) and oestradiol-17β (E2) levels in spent media were determined by ELISA. The expression of luteinization-related genes STAR (steroidogenic acute regulatory protein), HSD3B1 (3β-hydroxysteroid dehydrogenase type 1), PGR (genomic P4 receptor), and PGRMC1 and PGRMC2 (P4 receptor membrane components 1 and 2) was analysed by real-time RT-PCR. The data from 6 to 8 replicates (at least 170 oocytes for each condition) were analysed by ANOVA following by Tukey’s (steroidogenesis) or Dunn’s (gene expression) tests. In the control group, the level of P4 production exhibited by COC during 24-h aging was 5.5-fold higher than the level observed during in vitro maturation (1.50 ± 0.15 ng v. 0.27 ± 0.02 ng of P4/COC; P &lt; 0.001). Conversely, E2 production by aging COC was 2.7 times lower than that by maturing COC (0.39 ± 0.05 pg v. 1.04 ± 0.05 pg of E2/COC; P &lt; 0.001). The CC expression of genes responsible for P4 synthesis, STAR and HSD3B1, increased progressively by 24 h of aging (7.1- and 34.6-fold, respectively; P &lt; 0.001). Meanwhile, the steroid secretion and expression of STAR and HSD3B1 genes were unaffected by PRL. The relative mRNA abundance of PGRMC1 increased 2.3-fold (P &lt; 0.05) in the control (at 24 h) and PRL groups (at 12 h), while that of PGRMC2 did not change significantly in both groups. In addition, PGR transcript level decreased (P &lt; 0.01) 9.9-fold at 12 h and 18.3-fold at 24 h aging of PRL-treated COC, whereas this decrease was only 5-fold (P &lt; 0.05) at 24 h in Control. The findings indicate that aging of bovine oocytes matured in vitro enhances luteinization of surrounding CC. Prolactin does not affect steroidogenesis but may modulate the expression of P4 receptors in cumulus cells. This research was supported partly by FASO Russia and INRA (France).

Progesterone-Induced miR-152 Inhibits the Proliferation of Endometrial Epithelial Cells by Downregulating WNT-1.

Progesterone (P4) is an important ovarian hormone that inhibits estrogen-dependent proliferation of endometrial epithelial cells (EECs). miR-152 has been reported to be a cell cycle regulator. In this study, we first demonstrated that P4 induced the expression of miR-152 in ovariectomized mice and Ishikawa cell. miR-152 was detected in the human endometrial cell lines that were stably transfected with P4 receptor. Results showed that P4 induced its expression through its receptor B subtype. Then, using the specific miRNA mimic and inhibitor, we proved that miR-152 impeded G1/S transition in the cell cycle of EECs and inhibited cellular proliferation via downregulating WNT-1 in mice and human endometrial cancer cell lines (Ishikawa, HEC-1-b, and KLE). miR-152 induced by P4 is an important inhibitor for the proliferation of EECs. miR-152 may be an important tumor suppressor microRNA in endometrial cancer.

Dynamic expression of PGRMC1 and SERBP1 in human endometrium: an implication in the human decidualization process

To characterize PGRMC1 and SERBP1 in human endometrium and to investigate the putative role of PGRMC1 in endometrial decidualization. The PGRMC1 and SERBP1 expression in human endometrium was determined throughout the menstrual cycle. We analyzed the colocalization of PGRMC1 and SERBP1. Then, endometrial stromal cells (ESCs) were isolated to investigate the functional effect of PGRMC1 overexpression on decidualization. IVI clinic. Endometrial biopsies were collected from fertile volunteers (n = 61) attending the clinic as ovum donors. Endometrial samples of 61 healthy fertile women. Invivo localization of PGRMC1 and SERBP1 was assessed by immunohistochemistry. The PGRMC1/SERBP1 colocalization was investigated invitro and invivo. Decidualization effect of PGRMC1 overexpression was evaluated in primary ESC cultures. The PGRMC1 was detected in the endometrial stroma throughout the menstrual cycle, but decreased in the late secretory phase. The SERBP1 immunostaining was present in stroma and increased in the entire the menstrual cycle. The PGRMC1 and SERBP1 colocalized in the cytoplasmic fractions of nondecidualized and decidualized ESC. The PGRMC1 overexpression significantly inhibited invitro decidualization. Our results suggest that classic P receptors (PRs) are not the only kind playing a role in the normal physiology of the endometrium. The human decidualization process could be altered by the overexpression or mislocalization of PGRMC1 in ESC.

Abstract DPOC-007: PROGESTERONE/PR ERADICATES p53–DEFECTIVE CELLS IN THE FALLOPIAN TUBE EPITHELIUM BY INDUCTION OF NECROPTOSIS

Abstract Cervical Cancer Prevention Center, Department of Research, Tzu Chi Medical Center, Hualien, Taiwan, Republic of China. Institute of Medical Sciences, Tzu Chi University, Taiwan, Republic of China. The fallopian tube (FT) fimbriae, which are regularly exposed to ovulatory carcinogens, have been identified as the origin of high–grade serous ovarian carcinoma (HGSOC), with loss of p53 determined to be a universal and the earliest step of carcinogenesis. In previous studies, we have identified ROS in ovarian follicle is highly mutagenic and toxic to the fallopian tube fimbria that bath in the ovulatory follicular fluid (FF) after ovulation. We also found that loss of p53 renders fimbrial epithelial cells less vulnerable to the ROS stress, and that hemoglobin in ovulated FF could rescue the ROS–stressed fimbrial epithelial cells from apoptosis. Given that both ovulation and retrograde menstruation, the two proven risk factors for HGSOC, are commonplace in menstrual cycles, and that occurrence of tubal p53 signature is frequent but HGSOC is rare, we hypothesize that luteal–phase progesterone (P4) is responsible for clearance of p53–defective epithelial lesions as a natural protection. Herein, we show that P4 and progesterone receptor (PR) induce necroptosis of tubal epithelial cells in p53–null mice and in immortalized p53–deficient human fimbria epithelial cells through the TNF–α/RIPK1/RIPK3/p–MLKL pathway. The clearance acts regularly in the luteal phase of the menstrual cycle, recovering shortly thereafter, and can be augmented with P4 supplementation. With the inhibition of PR, the oviduct epithelium of Trp53–null mice escapes the clearance and, upon repeated ovulation exposures, accumulates DNA double–strand breaks. Through the induction of necroptosis of p53–deficient epithelial cells, luteal phase P4 prevents the transformation of the FT epithelium after ovulation. This protection can be augmented with P4 supplementation. Citation Format: Tang–Yuan Chu, MD, PhD. PROGESTERONE/PR ERADICATES p53–DEFECTIVE CELLS IN THE FALLOPIAN TUBE EPITHELIUM BY INDUCTION OF NECROPTOSIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-007.

Progesterone Prevents High-Grade Serous Ovarian Cancer by Inducing Necroptosis of p53-Defective Fallopian Tube Epithelial Cells

High-grade serous ovarian carcinoma (HGSOC) originates mainly from the fallopian tube (FT) epithelium and always carries early TP53 mutations. We previously reported that tumors initiate in the FT fimbria epithelium because of apoptotic failure and the expansion of cells with DNA double-strand breaks (DSB) caused by bathing of the FT epithelial cells in reactive oxygen species (ROSs) and hemoglobin-rich follicular fluid (FF) after ovulation. Because ovulation is frequent and HGSOC is rare, we hypothesized that luteal-phase progesterone (P4) could eliminate p53-defective FT cells. Here we show that P4, via P4 receptors (PRs), induces necroptosis in Trp53-/- mouse oviduct epithelium and in immortalized human p53-defective fimbrial epithelium through the TNF-α/RIPK1/RIPK3/MLKL pathway. Necroptosis occurs specifically at diestrus, recovers at the proestrus phase of the estrus cycle, and can be augmented with P4 supplementation. These results reveal the mechanism of the well-known ability of progesterone to prevent ovarian cancer.

Effect of early pregnancy on the expression of progesterone receptor and progesterone-induced blocking factor in ovine lymph node

Lymph nodes are the sites where the immune reaction or suppression takes place. Progesterone (P4) exerts an essential effect of the immunomodulation on the maternal uterus during early pregnancy in ruminants. At present study, the inguinal lymph nodes were obtained at day 16 of non-pregnancy, days 13, 16 and 25 of pregnancy (n = 3 for each group) in ewes, and RT-PCR assay, western blot and immunohistochemistry analysis were used to analyze to the effect of early pregnancy on the expression of P4 receptor (PGR) and progesterone-induced blocking factor (PIBF) in the lymph nodes. Our results showed that the PGR and PIBF mRNA were up-regulated in the lymph nodes in pregnant ewes, and the PGR isoform (60 kDa) and the PIBF variant (75 kDa) were expressed constantly in the lymph nodes. However, there was no expression of the PGR isoform (40 kDa) and the PIBF variant (48 kDa) at day 16 of the estrous cycle. The immunohistochemistry results confirmed that the PGR and PIBF proteins were limited to the subcapsular sinus and trabeculae in the cortex, medullary sinuses, and were localized in the cytoplasm of the specific cells. This paper reports for the first time that early pregnancy exerts its effect on the specific cells in the lymph nodes through P4, which results in the up-regulated expression of the PGR mRNA and 40 kDa isoform, the PIBF mRNA and 48 kDa variant, and is involved in the immunoregulation of the lymph nodes through a cytosolic pathway in ewes.

Progesterone Protects Against Bisphenol A-Induced Arrhythmias in Female Rat Cardiac Myocytes via Rapid Signaling.

Bisphenol A (BPA) is an estrogenic endocrine-disrupting chemical (EDC) that has a range of potential adverse health effects. Previously we showed that acute exposure to BPA promoted arrhythmias in female rat hearts through estrogen receptor rapid signaling. Progesterone (P4) and estrogen have antagonistic or complementary actions in a number of tissues and systems. In the current study, we examined the influence and possible protective effect of P4 on the rapid cardiac actions of BPA in female rat cardiac myocytes. Preincubation with physiological concentration (1 nM) of P4 abolished BPA-induced triggered activities in female cardiac myocytes. Further, P4 abrogated BPA-induced alterations in Ca2+ handling, including elevated sarcoplasmic reticulum Ca2+ leak and Ca2+ load. Key to the inhibitory effect of P4 is its blockade of BPA-induced increase in the phosphorylation of phospholamban. At myocyte and protein levels, these inhibitory actions of P4 were blocked by pretreatment with the nuclear P4 receptor (nPR) antagonist RU486. Analysis using membrane-impermeable bovine serum albumin-conjugated P4 suggested that the actions of P4 were mediated by membrane-initiated signaling. Inhibitory G (Gi) protein and phophoinositide-3 kinase (PI3K), but not tyrosine protein kinase activation, were involved in the observed effects of P4. In conclusion, P4 exerts an acute protective effect against BPA-induced arrhythmogenesis in female cardiac myocytes through nPR and the Gi/PI3K signaling pathway. Our findings highlight the importance of considering the impact of EDCs in the context of native hormonals and may provide potential therapeutic strategies for protection against the cardiac toxicities associated with BPA exposure.

Neurosteroids and Ischemic Stroke: Progesterone a Promising Agent in Reducing the Brain Injury in Ischemic Stroke.

Progesterone (P4), a well-known neurosteroid, is produced by ovaries and placenta in females and by adrenal glands in both sexes. Progesterone is also synthesized by central nervous system (CNS) tissues to perform various vital neurological functions in the brain. Apart from performing crucial reproductive functions, it also plays a pivotal role in neurogenesis, regeneration, cognition, mood, inflammation, and myelination in the CNS. A substantial body of experimental evidence from animal models documents the neuroprotective role of P4 in various CNS injury models, including ischemic stroke. Extensive data have revealed that P4 elicits neuroprotection through multiple mechanisms and systems in an integrated manner to prevent neuronal and glial damage, thus reducing mortality and morbidity. Progesterone has been described as safe for use at the clinical level through different routes in several studies. Data regarding the neuroprotective role of P4 in ischemic stroke are of great interest due to their potential clinical implications. In this review, we succinctly discuss the biosynthesis of P4 and distribution of P4 receptors (PRs) in the brain. We summarize our work on the general mechanisms of P4 mediated via the modulation of different PR and neurotransmitters. Finally, we describe the neuroprotective mechanisms of P4 in ischemic stroke models and related clinical prospects.

Progesterone Receptor Membrane Component 1 Mediates Progesterone-Induced Suppression of Oocyte Meiotic Prophase I and Primordial Folliculogenesis.

Well-timed progression of primordial folliculogenesis is essential for mammalian female fertility. Progesterone (P4) inhibits primordial follicle formation under physiological conditions; however, P4 receptor that mediates this effect and its underlying mechanisms are unclear. In this study, we used an in vitro organ culture system to show that progesterone receptor membrane component 1 (PGRMC1) mediated P4-induced inhibition of oocyte meiotic prophase I and primordial follicle formation. We found that membrane-impermeable BSA-conjugated P4 inhibited primordial follicle formation similar to that by P4. Interestingly, PGRMC1 and its partner serpine1 mRNA-binding protein 1 were highly expressed in oocytes in perinatal ovaries. Inhibition or RNA interference of PGRMC1 abolished the suppressive effect of P4 on follicle formation. Furthermore, P4-PGRMC1 interaction blocked oocyte meiotic progression and decreased intra-oocyte cyclic AMP (cAMP) levels in perinatal ovaries. cAMP analog dibutyryl cAMP reversed P4–PGRMC1 interaction-induced inhibition of meiotic progression and follicle formation. Thus, our results indicated that PGRMC1 mediated P4-induced suppression of oocyte meiotic progression and primordial folliculogenesis by decreasing intra-oocyte cAMP levels.

Progesterone Receptor-Mediated Actions Regulate Remodeling of the Cervix in Preparation for Preterm Parturition.

This study determined whether a progesterone (P) receptor (PR)-mediated mechanism regulates morphological characteristics associated with prepartum cervix remodeling at term and with preterm birth. With focus on the transition from a soft to ripe cervix, the cervix stroma of untreated controls had reduced cell nuclei density/area and less organized extracellular collagen, while the density of macrophages/area, but not neutrophils, increased just 2 days before birth (day 17 vs day 15 or 16.5 postbreeding). Preterm birth was induced within 24 hours of treatment on day 16 postbreeding with PR antagonist or ovariectomy (Ovx). Pure or mixed PR antagonists increased the density of macrophages in the cervix within 8 hours (day 16.5 postbreeding), in advance of preterm birth. However, neither PR antagonists nor P withdrawal after Ovx affected the densities of cell nuclei and neutrophils or extracellular collagen compared to the same day controls-an indication that the cervix was sufficiently remodeled for birth to occur. To block the effect of systemic P withdrawal, Ovx pregnant mice were given a PR agonist, either pure or mixed. These treatments forestalled preterm birth and prevented further morphological remodeling of the cervix. The resulting increase in macrophage density in cervix stroma following Ovx was only blocked by a pure PR agonist. These findings support the hypothesis that inflammatory processes in the prepartum cervix that include residency of macrophages, cellular hypertrophy, and extracellular collagen structure are regulated by genomic actions of PR in a final common mechanism both at term and with induced preterm birth.

Progestin-mediated activation of MAPK and AKT in nuclear progesterone receptor negative breast epithelial cells: The role of membrane progesterone receptors

Progesterone (P4), a steroid produced during estrous cycles and gestation for maintenance of pregnancy, also plays key roles in breast development to allow lactation post-parturition. Progestins (P4 and related steroids) are also implicated in breast cancer etiology. Hormone replacement therapy containing both estrogen and progestins increases breast cancer incidence while estrogen hormone therapy lowers breast cancer risk. P4 signaling via nuclear P4 receptors (PRs) has been extensively studied in breast cancer, however, progestin signaling via non-classical membrane bound progestin receptors (MPRs and PGRMC1) remains unclear. Moreover, P4 metabolites and synthetic progestins may bind membrane progestin receptors. We hypothesized that PR-negative breast epithelial cells express non-classical progestin receptors, which activate intracellular signaling pathways differently depending on nature of progestin. Therefore, our objectives for the current study were to determine expression of MPRs and PGRMC1 in two PR-negative non-tumorigenic breast epithelial cell lines, assess progestin-mediated signaling and biological functions. We determined five MPR isoforms and PGRMC1 were present in MCF10A cells and all progestin receptors but MPRβ in MCF12A cells. MCF10A and MCF12A cells were treated with P4, select P4 metabolites (5αP and 3αHP), medroxyprogesterone acetate (MPA), or a specific MPR-Agonist (MPR-Ag) and phosphorylation of ERK, p38, JNK, and AKT was characterized following treatment. To our knowledge this is the first report of ERK and JNK activation in MCF10A and MCF12A cells with P4, P4 metabolites, MPA, and MPR-Ag. Activation of ERK and JNK in cells treated with MPR-Ag implicates MPRs may serve as the receptors responsible for their activation. In contrast, p38 activation varied with cell type and with progestin treatment. P4 and MPA promoted AKT phosphorylation in the MCF12A cell line only whereas no activation was observed in MCF10A cells. Interestingly, cellular proliferation increased in MCF10A cells treated with MPA or 5αP, while MPR-Ag tended to slightly decrease proliferation. Collectively, our data highlights the importance of investigating the effects of synthetic progestins in breast cancer biology. Our results add to the understanding that various progestins have on breast epithelial cells and underscores the importance of considering both membrane bound receptors and progestin type in breast cancer development.

P-glycoprotein expression and localization in the rat uterus throughout gestation and labor.

Uterine tissues contain the efflux transporter P-glycoprotein (P-gp, encoded by Abcb1a/1b gene), but little is known about how it changes through gestation. Our aim was to investigate the expression profile and cellular localization of P-gp in the pregnant, laboring and post-partum (PP) rat uterus. We propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial Abcb1a/1b/P-gp. Samples from bilaterally and unilaterally pregnant rats were collected throughout gestation, during labor, and PP (n=4-6/gestational day). RNA and protein were isolated and subjected to quantitative PCR and immunoblotting; P-gp transcript and protein were localized by in situ hybridization and immunohistochemistry. Expression of Abcb1a/1b gene and membrane P-gp protein in uterine tissue (1) increased throughout gestation, peaked at term (GD19-21) and dropped during labor (GD23L); and (2) was upregulated only in gravid but not in empty horn of unilaterally pregnant rats. (3) The drop of Abcb1a/1b mRNA on GD23 was prevented by artificial maintenance of elevated progesterone (P4) levels in late gestation; (4) injection of the P4 receptor antagonist RU486 on GD19 caused a significant decrease in Abcb1 mRNA levels. (5) In situ hybridization and immunohistochemistry indicated that Abcb1/P-gp is absent from myometrium throughout gestation; (6) was expressed exclusively by uterine microvascular endothelium (at early gestation) and luminal epithelium (at mid and late gestation), but was undetectable during labor. In conclusion, ABC transporter protein P-gp in pregnant uterus is hormonally and mechanically regulated. However, its substrate(s) and precise function in these tissues during pregnancy remains to be determined.