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Objective: To determine the relationship between progesterone receptor membrane component-1 (PGRMC1) expression and the outcome of IVF treatment.Design: A prospective study in which PGRMC1 messenger RNA (mRNA) levels, methylation status of the Pgrmc1 promoter, and the presence of point mutations within Pgrmc1 were obtained from granulosa (GC)/luteal cells of women undergoing controlled ovarian hyperstimulation (COH).Setting: Fertility center/basic science laboratory.Patient(s): Eighty-five patients undergoing IVF treatment and 10 women who were undergoing COH for the purpose of oocyte donation were included in this study.Intervention(s): None.Main Outcome Measure(s): The PGRMC1 measurements were correlated with clinical outcomes, such as number of follicles, number of retrieved oocytes, and ongoing pregnancy rates (PR).Result(s): The PGRMC1 mRNA levels within GC/luteal cells of 18% of IVF patients were >2.25-fold higher than those of oocyte donors. Individuals with elevated PGRMC1 mRNA levels had 30% fewer large follicles and fewer oocytes retrieved. The elevated PGRMC1 mRNA levels were associated with an increase in the methylation of Pgrmc1 promoter.Conclusion(s): In patients with elevated PGRMC1 mRNA levels, gonadotropin-induced follicle development is attenuated, although sufficient numbers of follicles develop to allow for ET and subsequent pregnancy. [ABSTRACT FROM AUTHOR]

Purpose: This study was designed to determine if DMO limits in vitro development of aneuploid-enriched mouse embryos by activating a Trp53-dependent mechanism., Methods: Mouse cleavage-stage embryos were treated with reversine to induce aneuploidy or vehicle to generate controls, and then cultured in media supplemented with DMO to reduce the pH of the culture media. Embryo morphology was assessed by phase microscopy. Cell number, mitotic figures, and apoptotic bodies were revealed by staining fixed embryos with DAPI. mRNA levels of Trp53, Oct-4, and Cdx2 were monitored by quantitative polymerase chain reactions (qPCRs). The effect of Trp53 on the expression of Oct-4 and Cdx2 was assessed by depleting Trp53 using Trp53 siRNA., Results: Aneuploid-enriched late-stage blastocysts were morphologically indistinguishable from control blastocysts but had fewer cells and reduced mRNA levels of Oct-4 and Cdx2. Adding 1 mM DMO to the culture media during the 8-cell to blastocyst transition reduced the formation of aneuploid-enriched late-stage blastocysts but not control blastocysts and further suppressed the levels of Oct-4 and Cdx2 mRNA. Trp53 RNA levels in aneuploid-enriched embryos that were exposed to DMO were > twofold higher than controls, and Trp53 siRNA levels reduced the levels of Trp53 and increased levels of Oct-4 and Cdx2 mRNA by > twofold., Conclusion: These studies suggest that the development of morphologically normal aneuploid-enriched mouse blastocysts can be inhibited by adding low amounts of DMO to the culture media, which results in elevated levels of Trp53 mRNA that suppresses Oct-4 and Cdx2 expression., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To determine the relationship between the levels of cumulus cell (CC) hemoglobin messenger ribonucleic acid (mRNA) and the developmental potential of the associated oocyte and whether hemoglobin protects the CCs from oxidative stress-induced apoptosis., Design: Laboratory-based study., Setting: University laboratory and university-affiliated in vitro fertilization center., Patient(s): Cumulus cells from the oocytes of patients who underwent in vitro fertilization with intracytoplasmic sperm injection with and without preimplantation genetic testing between 2018 and 2020., Intervention(s): Studies on individual and pooled CCs collected at the time of oocyte retrieval or cultured under 20% or 5% O 2 ., Main Outcome Measure(s): Quantitative polymerase chain reaction analysis of individual and pooled patient CC samples were used to monitor the hemoglobin mRNA levels. Reverse transcription-polymerase chain reaction arrays were used to assess genes that regulate oxidative stress in CCs associated with aneuploid and euploid blastocysts. Studies were conducted to assess the effect of oxidative stress on the rate of apoptosis, level of reactive oxygen species, and gene expression in CCs in vitro., Result(s): Compared with CCs associated with arrested and aneuploid blastocysts, the mRNA levels encoding the alpha and beta chains of hemoglobin increased by 2.9- and 2.3-fold in CCs associated with euploid blastocysts, respectively. The mRNA levels encoding the alpha and beta chains of hemoglobin also increased by 3.8- and 4.5-fold in CCs cultured under 5% O 2 vs. 20% O 2 , respectively, and multiple regulators of oxidative stress were overexpressed in cells cultured under 20% O 2 compared with those under 5% O 2 . However, the rate of apoptosis and amount of mitochondrial reactive oxidative species increased by 1.25-fold in CCs cultured under 20% O 2 compared with those under 5% O 2 . Variable amounts of the alpha and beta chains of hemoglobin were also detected within the zona pellucida and oocytes., Conclusion(s): Higher levels of nonerythroid hemoglobin in CCs are associated with oocytes that result in euploid blastocysts. Hemoglobin may protect CCs from oxidative stress-induced apoptosis, which may enhance cumulus-oocyte interactions. Moreover, CC-derived hemoglobin may be transferred to the oocytes and protect it from the adverse effects of oxidative stress that occurs in vivo and in vitro., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

How progesterone influences ovarian follicle growth is a difficult question to answer because ovarian cells synthesize progesterone and express not only the classic nuclear progesterone receptor but also members of the progestin and adipoQ receptor family and the progesterone receptor membrane component (PGRMC) family. Which type of progestin receptor is expressed depends on the ovarian cell type as well as the stage of the estrous/menstrual cycle. Given the complex nature of the mammalian ovary, this review will focus on progesterone signaling that is transduced by PGRMC1 and PGRMC2 specifically as it relates to ovarian follicle growth. PGRMC1 was identified as a progesterone binding protein cloned from porcine liver in 1996 and detected in the mammalian ovary in 2005. Subsequent studies focused on PGRMC family members as regulators of granulosa cell proliferation and survival, two physiological processes required for follicle development. This review will present evidence that demonstrates a causal relationship between PGRMC family members and the promotion of ovarian follicle growth. The mechanisms through which PGRMC-dependent signaling regulates granulosa cell proliferation and viability will also be discussed in order to provide a more complete understanding of our current concept of how progesterone regulates ovarian follicle growth.

Cancers of the female reproductive tract are both lethal and highly prevalent. For example, the five-year survival rate of women diagnosed with ovarian cancer is still less than 50%, and endometrial cancer is the fourth most common cancer in women with > 65,000 new cases in the United States in 2020. Among the many genes already established as key participants in ovarian and endometrial oncogenesis, progesterone receptor membrane component (PGRMC)1 and PGRMC2 have gained recent attention given that there is now solid correlative information supporting a role for at least PGRMC1 in enhancing tumor growth and chemoresistance. The expression of PGRMC1 is significantly increased in both ovarian and endometrial cancers, similar to that reported in other cancer types. Xenograft studies using human ovarian and endometrial cancer cell lines in immunocompromised mice demonstrate that reduced expression of PGRMC1 results in tumors that grow substantially slower. While the molecular underpinnings of PGRMCs' mechanisms of action are not clearly established, it is known that PGRMCs regulate survival pathways that attenuate stress-induced cell death. The objective of this review is to provide an overview of what is known about the roles that PGRMC1 and PGRMC2 play in ovarian and endometrial cancers, particularly as related to the mechanisms through which they regulate mitosis, apoptosis, chemoresistance, and cell migration.

During the last few decades, millions of healthy children have been born with the aid of in vitro fertilization (IVF). This success belies the fact that IVF treatment is comprised of a complex series of interventions starting with a customized control ovarian stimulation protocol. This is followed by the induction of oocyte maturation, the retrieval of mature oocytes and in vitro fertilization, which often involves the microinjection of a single sperm into the oocyte. After fertilization, the resulting embryos are cultured for up to 7 days. The best embryos are transferred into the uterus where the embryo implants and hopefully develops into a healthy child. However, frequently the best embryos are biopsied and frozen. The biopsied cells are analyzed to identify those embryos without chromosomal abnormalities. These embryos are eventually thawed and transferred with pregnancy rates as good if not better than embryos that are not biopsied and transferred in a fresh cycle. Thus, IVF treatment requires the coordinated efforts of physicians, nurses, molecular biologists and embryologists to conduct each of these multifaceted phases in a seamless and flawless manner. Even though complex, IVF treatment may seem routine today, but it was not always the case. In this review the evolution of human IVF is presented as a series of innovations that resolved a technical hurdle in one component of IVF while creating challenges that eventually lead to the next major advancement. This step-by-step evolution in the treatment of human infertility is recounted in this review., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

MicroRNA-21 is expressed in bovine, murine, and human cumulus cells with its expression in murine and bovine cumulus cells correlated with oocyte developmental potential. The aim of this study was to assess the relationship between cumulus cell MIR-21 and human oocyte developmental potential. These studies revealed that both the immature and mature forms of MicroRNA-21 (MIR-21-5p) were elevated in cumulus cells of oocytes that developed into blastocysts compared to cumulus cells of oocytes that arrested prior to blastocyst formation. This increase in MicroRNA-21 was observed regardless of whether the oocytes developed into euploid or aneuploid blastocysts. Moreover, MIR-21-5p levels in cumulus cells surrounding oocytes that either failed to mature or matured to metaphase II but failed to fertilize, were ≈50% less than the MIR-21-5p levels associated with oocytes that arrested prior to blastocyst formation. Why cumulus cells associated with oocytes of reduced developmental potential expressed less MIR-21-5p is unknown. It is unlikely due to reduced expression of either the receptors of growth differentiation factor 9 or rosha Ribonuclease III (DROSHA) and Dicer Ribonuclease III (DICER) which sequentially promote the conversion of immature forms of MicroRNA-21 to mature MicroRNA-21. Furthermore, cultured cumulus cells treated with a MIR-21-5p inhibitor had an increase in apoptosis and a corresponding increase in the expression of PTEN, a gene known to inhibit the AKT-dependent survival pathway in cumulus cells. These studies provide evidence for a role of MicroRNA-21 in human cumulus cells that influences the developmental potential of human oocytes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes 1,2 . The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown 3,4 . Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined 3,4 . Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

Progesterone receptor membrane component 1 (PGRMC1) interacts with PGRMC2, and disrupting this interaction in spontaneously immortalized granulosa cells (SIGCS) leads to an inappropriate entry into the cell cycle, mitotic arrest, and ultimately cell death. The present study revealed that PGRMC1 and PGRMC2 localize to the cytoplasm of murine granulosa cells of nonatretric follicles with their staining intensity being somewhat diminished in granulosa cells of atretic follicles. Compared to controls (Pgrmc1fl/fl), the rate at which granulosa cells entered the cell cycle increased in nonatretic and atretic follicles of mice in which Pgrmc1 was conditionally deleted (Pgrmc1d/d) from granulosa cells. This increased rate of entry into the cell cycle was associated with a ≥ 2-fold increase in follicular atresia and the nuclear localization of nuclear factor-kappa-B transcription factor P65; (NFΚB/p65, or RELA). GTPase activating protein binding protein 2 (G3BP2) binds NFΚB/p65 through an interaction with NFΚB inhibitor alpha (IκBα), thereby maintaining NFΚB/p65's cytoplasmic localization and restricting its transcriptional activity. Since PGRMC1 and PGRMC2 bind G3BP2, studies were designed to assess the functional relationship between PGRMC1, PGRMC2, and NFΚB/p65 in SIGCs. In these studies, disrupting the interaction between PGRMC1 and PGRMC2 increased the nuclear localization of NFΚB/p65, and depleting PGRMC1, PGRMC2, or G3BP2 increased NFΚB transcriptional activity and the progression into the cell cycle. Taken together, these studies suggest that PGRMC1 and 2 regulate granulosa cell cycle entry in follicles by precisely controlling the localization and thereby the transcriptional activity of NFΚB/p65., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

To determine whether conditional depletion of progesterone receptor membrane component (PGRMC) 1 and PGRMC2 affected ovarian follicle development, follicle distribution was assessed in ovaries of young (≈3-month-old) and middle-aged (≈6-month-old) control (Pgrmc1/2fl/fl) and double conditional PGRMC1/2-knockout (Pgrmc1/2d/d) mice. This study revealed that the distribution of primary, preantral and antral follicles was not altered in Pgrmc1/2d/d mice, regardless of the age. Although the number of primordial follicles was similar at ≈3 months of age, their numbers were reduced by ≈80% in 6-month-old Pgrmc1/2d/d mice compared to age-matched Pgrmc1/2fl/fl mice. The Pgrmc1/2d/d mice were generated using Pgr-cre mice, so ablation of Pgrmc1 and Pgrmc2 in the ovary was restricted to peri-ovulatory follicles and subsequent corpora lutea (CL). In addition, the vascularization of CL was attenuated in Pgrmc1/2d/d mice, although mRNA levels of vascular endothelial growth factor A (Vegfa) were elevated. Moreover, depletion of Pgrmc1 and Pgrmc2 altered the gene expression profile in the non-luteal component of the ovary such that Vegfa expression, a stimulator of primordial follicle growth, was elevated; Kit Ligand expression, another stimulator of primordial follicle growth, was suppressed and anti-Mullerian hormone, an inhibitor of primordial follicle growth, was enhanced compared to Pgrmc1/2fl/fl mice. These data reveal that luteal cell depletion of Pgrmc1 and 2 alters the expression of growth factors within the non-luteal component of the ovary, which could account for the premature demise of the adult population of primordial follicles. In summary, the survival of adult primordial follicles is dependent in part on progesterone receptor membrane component 1 and 2., (2018 Society for Reproduction and Fertility)

Progesterone receptor membrane component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1's nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi)-mediated gene silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H 2 O 2 ) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1's ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1's well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress., (© 2018 Society for Reproduction and Fertility.)

The present studies were designed to determine whether progesterone (P4)-progesterone receptor membrane component 1 (PGRMC1) signaling is able to attenuate the apoptotic effects of oxidative stress induced by hydrogen peroxide (H2O2). To achieve this goal, freshly isolated human granulosa/luteal cells were maintained in culture. After several passages, the cells were treated with H2O2, which induced apoptosis within 2.5 h, while simultaneous treatment with P4 attenuated the apoptotic action of H2O2. AG 205, a PGRMC1 antagonist, eliminated P4's ability to prevent H2O2-induced apoptosis. AG 205 neither affected PGRMC1's cytoplasmic localization nor its interaction with PGRMC2, but appeared to reduce its presence within the nucleus. AG 205 also (1) increased the monomeric and decreased the higher molecular weight forms of PGRMC1 (i.e., dimers/oligomers) and (2) altered the expression of several genes involved in apoptosis. The most dramatic change was an approximate 8-fold increase in Harakiri (Hrk) mRNA. However, AG 205 did not induce apoptosis in the absence of H2O2. Taken together, these observations suggest that the higher molecular weight forms of PGRMC1 likely account in part for PGRMC1's ability to suppress the expression of Hrk. Harakiri is a BH-3 only member of the B-cell lymphoma 2 (BCL2) family that promotes apoptosis by binding to and antagonizing the antiapoptotic action of BCL2- and BCL2-like proteins. It is likely then that PGRMC1's ability to suppress Hrk is part of the mechanism through which P4-PGRMC1 signaling preserves the viability of human granulosa/luteal cells., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The nonclassical progesterone receptors progesterone receptor membrane component (PGRMC) 1 and PGRMC2 have been implicated in regulating cell survival of endometrial and ovarian cells in vitro and are abundantly expressed in these cell types. The objective of this study was to determine if Pgrmc1 and Pgrmc2 are essential for normal female reproduction. To accomplish this objective, Pgrmc1 and/or Pgrmc2 floxed mice (Pgrmc2fl/fl and Pgrmc1/2fl/fl) were crossed with Pgr-cre mice, which resulted in the conditional ablation of Pgrmc1 and/or Pgrmc2 from female reproductive tissues (i.e.,Pgrmc2d/d and Pgrmc1/2d/d mice). A breeding trial revealed that conditional ablation of Pgrmc2 initially led to subfertility, with Pgrmc2d/d female mice producing 47% fewer pups/litter than Pgrmc2fl/fl mice (P = 0.001). Pgrmc2d/d mice subsequently underwent premature reproductive senescence by parities 2 to 5, producing 37.8% fewer litters overall during the trial compared with Pgrmc2fl/fl mice (P = 0.020). Similar results were observed with Pgrmc1/2d/d mice. Based on ovarian morphology and serum P4, the subfertility/infertility was not due to faulty ovulation or luteal insufficiency. Rather an analysis of midgestation implantation sites revealed that postimplantation embryonic death was the major cause of the subfertility/infertility. As with our previous report of Pgrmc1d/d mice, Pgrmc2d/d and Pgrmc1/2d/d mice developed endometrial cysts consistent with accelerated aging of this tissue. Given the timing of postimplantation embryonic demise, uterine decidualization may be disrupted in mice deficient in PGRMC2 or PGRMC1/2. Overall, this study revealed that Pgrmc1 and/or Pgrmc2 are required for the maintenance of uterine histoarchitecture and normal female reproductive lifespan., (Copyright © 2017 by the Endocrine Society.)

Progesterone (P4) is essential for female fertility. The objective of this study was to evaluate the functional requirement of the nonclassical P4 receptor (PGR), PGR membrane component 1, in regulating female fertility. To achieve this goal, the Pgrmc1 gene was floxed by insertion of loxP sites on each side of exon 2. Pgrmc1 floxed (Pgrmc1(fl/fl)) mice were crossed with Pgr(cre) or Amhr2(cre) mice to delete Pgrmc1 (Pgrmc1(d/d)) from the female reproductive tract. A 6-month breeding trial revealed that conditional ablation of Pgrmc1 with Pgr(cre/+) mice resulted in a 40% reduction (P = .0002) in the number of pups/litter. Neither the capacity to ovulate in response to gonadotropin treatment nor the expression of PGR and the estrogen receptor was altered in the uteri of Pgrmc1(d/d) mice compared with Pgrmc1(fl/fl) control mice. Although conditional ablation of Pgrmc1 from mesenchymal tissue using Amhr2(cre/+) mice did not reduce the number of pups/litter, the total number of litters born in the 6-month breeding trial was significantly decreased (P = .041). In addition to subfertility, conditional ablation of Pgrmc1 using either Amhr2(cre/+) or Pgr(cre/+) mice resulted in the development of endometrial cysts starting around 4 months of age. Interestingly, pregnancy attenuated the formation of these uterine cysts. These new findings demonstrate that PGR membrane component 1 plays an important role in female fertility and uterine tissue homeostasis.

Triple negative breast cancers (TNBCs) are highly aggressive and grow in response to sex steroid hormones despite lacking expression of the classical estrogen (E2) and progesterone (P4) receptors. Since P4 receptor membrane component 1 (PGRMC1) is expressed in breast cancer tumors and is known to mediate P4-induced cell survival, this study was designed to determine the expression of PGRMC1 in TNBC tumors and the involvement of PGRMC1 in regulating proliferation and survival of TNBC cells in vitro and the growth of TNBC tumors in vivo. For the latter studies, the MDA-MB-231 (MDA) cell line derived from TNBC was used. These cells express PGRMC1 but lack expression of the classical P4 receptor. A lentiviral-based shRNA approach was used to generate a stably transfected PGRMC1-deplete MDA line for comparison to the PGRMC1-intact MDA line. The present studies demonstrate that PGRMC1: 1) is expressed in TNBC cells; 2) mediates the ability of P4 to suppress TNBC cell mitosis in vitro; 3) is required for P4 to reduce the apoptotic effects of doxorubicin in vitro; and 4) facilitates TNBC tumor formation and growth in vivo. Taken together, these findings indicate that PGRMC1 plays an important role in regulating the growth and survival of TNBC cells in vitro and ultimately in the formation and development of these tumors in vivo. Thus, PGRMC1 may be a therapeutic target for TNBCs.

The present studies were designed to determine the role of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, progestin and adipoQ receptor 7 (PAQR7), and progesterone receptor (PGR) in mediating the antimitotic action of progesterone (P4) in human granulosa/luteal cells. For these studies granulosa/luteal cells of 10 women undergoing controlled ovarian hyperstimulation were isolated, maintained in culture, and depleted of PGRMC1, PGRMC2, PAQR7, or PGR by siRNA treatment. The rate of entry into the cell cycle was assessed using the FUCCI cell cycle sensor to determine the percentage of cells in the G1/S stage of the cell cycle. PGRMC1, PGRMC2, PAQR7, and PGR mRNA levels were assessed by real-time PCR and their interactions monitored by in situ proximity ligation assays (PLAs). These studies revealed that PGRMC1, PGRMC2, PAQR7, and PGR were expressed by granulosa/luteal cells from all patients, with PGRMC1 mRNA being most abundant, followed by PAQR7, PGRMC2, and PGR. However, their mRNA levels showed considerable patient variation. P4's ability to suppress entry into the cell cycle was dependent on PGRMC1, PGRMC2, and PAQR7 but not PGR. Moreover, PLAs indicated that PGRMC1, PGRMC2, and PAQR7 formed a complex within the cytoplasm. Based on these studies, it is proposed that these three P4 mediators form a complex within the cytoplasm that is required for P4's action. Moreover, P4's ability to regulate human follicle development may be dependent in part on the expression levels of each of these P4 mediators., (© 2015 by the Society for the Study of Reproduction, Inc.)

Females may display dramatically different behavior depending on their state of ovulation. This is thought to occur through sex-specific hormones acting on behavioral centers in the brain. Whether incoming sensory activity also differs across the ovulation cycle to alter behavior has not been investigated. Here, we show that female mouse vomeronasal sensory neurons (VSNs) are temporarily and specifically rendered "blind" to a subset of male-emitted pheromone ligands during diestrus yet fully detect and respond to the same ligands during estrus. VSN silencing occurs through the action of the female sex-steroid progesterone. Not all VSNs are targeted for silencing; those detecting cat ligands remain continuously active irrespective of the estrous state. We identify the signaling components that account for the capacity of progesterone to target specific subsets of male-pheromone responsive neurons for inactivation. These findings indicate that internal physiology can selectively and directly modulate sensory input to produce state-specific behavior. PAPERCLIP., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Endometrial cancer is the leading gynecologic cancer in women in the United States with 52,630 women predicted to be diagnosed with the disease in 2014. The objective of this study was to determine if progesterone (P4) receptor membrane component 1 (PGRMC1) influenced endometrial cancer cell viability in response to chemotherapy in vitro and in vivo. A lentiviral-based shRNA knockdown approach was used to generate stable PGRMC1-intact and PGRMC1-deplete Ishikawa endometrial cancer cell lines that also lacked expression of the classical progesterone receptor (PGR). Progesterone treatment inhibited mitosis of PGRMC1-intact, but not PGRMC1-deplete cells, suggesting that PGRMC1 mediates the anti-mitotic actions of P4. To test the hypothesis that PGRMC1 attenuates chemotherapy-induced apoptosis, PGRMC1-intact and PGRMC1-deplete cells were treated in vitro with vehicle, P4 (1 µM), doxorubicin (Dox, 2 µg/ml), or P4 + Dox for 48 h. Doxorubicin treatment of PGRMC1-intact cells resulted in a significant increase in cell death; however, co-treatment with P4 significantly attenuated Dox-induced cell death. This response to P4 was lost in PGRMC1-deplete cells. To extend these observations in vivo, a xenograft model was employed where PGRMC1-intact and PGRMC1-deplete endometrial tumors were generated following subcutaneous and intraperitoneal inoculation of immunocompromised NOD/SCID and nude mice, respectively. Tumors derived from PGRMC1-deplete cells grew slower than tumors from PGRMC1-intact cells. Mice harboring endometrial tumors were then given three treatments of vehicle (1:1 cremophor EL: ethanol + 0.9% saline) or chemotherapy [Paclitaxel (15 mg/kg, i.p.) followed after an interval of 30 minutes by CARBOplatin (50 mg/kg)] at five day intervals. In response to chemotherapy, tumor volume decreased approximately four-fold more in PGRMC1-deplete tumors when compared with PGRMC1-intact control tumors, suggesting that PGRMC1 promotes tumor cell viability during chemotherapeutic stress. In sum, these in vitro and in vivo findings demonstrate that PGRMC1 plays a prominent role in the growth and chemoresistance of human endometrial tumors., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 are expressed in rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs) but their biological roles are not well defined. The present studies demonstrate that depleting either Pgrmc1 or Pgrmc2 in SIGCs increases entry into the cell cycle but does not increase cell proliferation. Rather, PGRMC1 and/or PGRMC2-deplete cells accumulate in metaphase and undergo apoptosis. Because both PGRMC1 and PGRMC2 localize to the mitotic spindle, their absence likely accounts for cells arresting in metaphase. Moreover, pull-down assays, colocalization studies and in situ proximity ligation assays (PLA) indicate that PGRMC1 binds PGRMC2. Disrupting the PGRMC1:PGRMC2 complex through the use of siRNA or the cytoplasmic delivery of a PGRMC2 antibody increases entry into the cell cycle. Conversely, overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion protein inhibits entry into the cell cycle. Subsequent studies reveal that depleting PGRMC1 and/or PGRMC2 reduces the percentage of cells in G0 and increases the percentage of cells in G1. These observations indicate that in addition to their role at metaphase, PGRMC1 and PGRMC2 are involved in regulating entry into the G1 stage of the cell cycle. Interestingly, both PGRMC1 and PGRMC2 bind GTPase-activating protein-binding protein 2 (G3BP2) as demonstrated by pull-down assays, colocalization assays, and PLAs. G3bp2 siRNA treatment also promotes entry into the G1 stage. This implies that dynamic changes in the interaction among PGRMC1, PGRMC2, and G3BP2 play an important protein regulating the rate at which SIGCs enter into the cell cycle., (© 2014 by the Society for the Study of Reproduction, Inc.)

Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development., (© 2014 by the Society for the Study of Reproduction, Inc.)

It has been known for over 3 decades that progesterone (P4) suppresses follicle growth. It has been assumed that P4 acts directly on granulosa cells of developing follicles to slow their development, as P4 inhibits both mitosis and apoptosis of cultured granulosa cells. However, granulosa cells of developing follicles of mice, rats, monkeys, and humans do not express the A or B isoform of the classic nuclear receptor for P4 (PGR). By contrast, these granulosa cells express other P4 binding proteins, one of which is referred to as PGR membrane component 1 (PGRMC1). PGRMC1 specifically binds P4 with high affinity and mediates P4's anti-mitotic and anti-apoptotic action as evidenced by the lack of these P4-dependent effects in PGRMC1-depleted cells. In addition, mice in which PGRMC1 is conditionally depleted in granulosa cells show diminished follicle development. While the mechanism through which P4 activation of PGRMC1 affects granulosa cell function is not well defined, it appears that PGRMC1 controls granulosa cell function in part by regulating gene expression in T-cell-specific transcription factor/lymphoid enhancer factor-dependent manner. Clinically, altered PGRMC1 expression has been correlated with premature ovarian failure/insufficiency, polycystic ovarian syndrome, and infertility. These collective studies provide strong evidence that PGRMC1 functions as a receptor for P4 in granulosa cells and that altered expression results in compromised reproductive capacity. Ongoing studies seek to define the components of the signal transduction cascade through which P4 activation of PGRMC1 results in the regulation of granulosa cell function.

Progesterone (P4) is synthesized in the ovary and acts directly on granulosa cells of developing ovarian follicles to suppress their rate of mitosis and apoptosis. Granulosa cells do not express nuclear progesterone receptor (PGR) but rather progesterone receptor membrane component-1 (PGRMC1). PGRMC1 binds P4 and mediates P4's actions, as evidenced by PGRMC1 siRNA studies. PGRMC1 acts by binding plasminogen activator inhibitor 1 RNA-binding protein and regulating gene expression. Specifically, PGRMC1 suppresses some genes that promote cell death (i.e., Bad, Caspase-3, Caspase-4). P4 regulates gene expression in part by inhibiting PGRMC1 binding to Tcf/Lef transcription sites, thereby reducing Tcf/Lef transcriptional activity. Since Tcf/Lef transcription sites are located within the promoters of genes that initiate mitosis and/or apoptosis (i.e., c-jun and c-myc), P4-PGRMC1 mediated suppression of these Tcf/Lef regulated genes could account for P4's actions. PGRMC1 expression is also altered in women with polycystic ovarian syndrome, premature ovarian failure and infertility. Collectively, these observations support a role for PGRMC1 in regulating human ovarian follicle development.

Oocytes isolated from cows of reproductive age with reduced antral follicle counts (AFC) have a diminished capacity of embryonic development, which may be related to alterations in the mechanism that directs the proper segregation of chromosomes. Because we demonstrated that progesterone receptor membrane component 1 (PGRMC1) is involved in chromosome congression and metaphase II (MII) plate formation, the present study was designed to determine 1) if the decrease in oocyte developmental competence observed in dairy cows with a reduced AFC is due to a higher incidence of aneuploidy and 2) whether alterations in PGRMC1 contributes to the incidence of aneuploidy. Oocytes from ovaries with reduced AFC and age-matched controls were matured in vitro and the occurrence of aneuploidy determined as well as the mRNA level and localization of PGRMC1. Although oocytes from ovaries with reduced AFC were capable of undergoing meiosis in vitro, these oocytes showed a 3-fold increase in aneuploidy compared to oocytes isolated from control ovaries (P < 0.05). Although Pgrmc1 mRNA levels were not altered, PGRMC1 and aurora kinase B (AURKB) failed to localize to precise focal points on MII chromosomes of oocytes from ovaries with reduced AFC. Furthermore, when oocytes of control ovaries were cultured with an inhibitor of AURKB activity, their MII plate was disrupted and PGRMC1 was not properly localized to the chromosomes. These results suggest that alterations in PGRMC1 and/or AURKB localization account in part for the increased aneuploidy and low development competence of oocytes from ovaries with reduced AFC.

Progesterone receptor membrane component 1 (PGRMC1) mediates the antiapoptotic action of progesterone (P4). PGRMC1 interacts with plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1), but the functional significance of this interaction is unknown. To examine the function of PGRMC1-PAIRBP1 interaction, PAIRBP1 was depleted from spontaneously immortalized granulosa cells (SIGCs) and the effects on the expression and localization of PGRMC1 as well as P4's ability to bind to SIGCs and prevent apoptosis was assessed. Depleting PAIRBP1 enhanced cellular (3)H-P4 binding and did not alter the expression or cellular localization of PGRMC1 but attenuated P4's antiapoptotic action. Transfection of a PGRMC1-green fluorescent protein (GFP) peptide mimic, which binds PAIRBP1 as demonstrated by in situ proximity assay, doubled the rate at which SIGCs undergo apoptosis compared to cells transfected with either the empty GFP expression vector or Pairbp1 small interfering RNA. Moreover, P4 did not prevent these cells from undergoing apoptosis. Similar studies conducted with granulosa cells isolated from immature rats also showed that PGRMC1 interacts with PAIRBP1 and that transfection of PGRMC1-GFP peptide mimic accelerates the rate of granulosa cell apoptosis by 4-fold even in the presence of serum and P4. These studies support the concept that the interaction between PAIRBP1-PGRMC1 is an essential component of the mechanism through which P4 inhibits apoptosis. Surprisingly, PGRMC1-PAIRBP1 interaction is not required for P4 binding or the cellular localization of PGRMC1 but rather appears to couple PGRMC1 to downstream components of the P4-PGRMC1 signal transduction pathway.

Progesterone (P4) receptor membrane component (PGRMC)1 is detected as a 22-kDa band as well as higher molecular mass bands (>50 kDa) in spontaneously immortalized granulosa cells. That these higher molecular mass bands represent PGRMC1 is supported by the findings that they are not detected when either the primary antibody is omitted or the PGRMC1 antibody is preabsorbed with recombinant PGRMC1. Some but not most of the higher molecular mass bands are due to oligomerization. At least one of the higher molecular mass bands is sumoylated, because PGRMC1 coimmunoprecipitates with small ubiquitin-like modifier protein-1. Moreover, in situ proximity ligation assays reveal a direct interaction between PGRMC1 and small ubiquitin-like modifier protein-1. This interaction is increased by P4. Finally, the higher molecular mass forms of PGRMC1 localize to the nucleus. An analysis of transcription factor activity demonstrates that P4 suppresses T-cell factor/lymphoid enhancer factor (Tcf/Lef) activity through a PGRMC1-dependent mechanism, because treatment with PGRMC1 small interfering RNA depletes PGRMC1 levels and attenuates P4's effects on Tcf/Lef activity. In addition, transfection of a PGRMC1-Flag fusion protein enhances basal Tcf/Lef activity, which is suppressed by P4 treatment. Conversely, transfection of a PGRMC1-Flag protein in which all the sumoylation sites are mutated increases basal Tcf/Lef activity but attenuates P4's ability to suppress Tcf/Lef activity. Therefore, the ability to suppress Tcf/Lef activity is likely an essential part of the mechanism through which P4 activation of PGRMC1 regulates the gene cascades that control granulosa cell function with this action being dependent in part on the sumoylation status of PGRMC1.

Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the granulosa and luteal cells of rodent and primate ovaries. Interestingly, its molecular weight as assessed by Western blot is dependent on its cellular localization with a ≈27kDa form being detected in the cytoplasm and higher molecular weight forms being detected in the nucleus. The higher molecular weight forms of PGRMC1 are sumoylated suggesting that they are involved in regulating gene transcription, since sumoylation of nuclear proteins often is associated with regulation of transcriptional activity of the sumoylated protein. In order to identify a set of candidate genes that are regulated by PGRMC1, a human granulosa/luteal cell line (hGL5 cells) was treated with PGRMC1 siRNA and changes in gene expression monitored by microarray analysis. The microarray analysis revealed that PGRMC1 generally functioned as a repressor of transcription, since depletion of PGRMC1 resulted in a disproportionate increase in the number of transcripts. Moreover, a pathway analysis implicated PGRMC1 in the regulation of apoptosis, which is consistent with PGRMC1's known biological action. More importantly these results support the concept that PGRMC1 influences gene transcription. Additional studies reveal that progesterone (P4) acting through a PGRMC1-dependent mechanism suppresses the activity of the transcription factor, Tcf/Lef, thereby identifying one molecular pathway through which P4-PGRMC1 can regulate gene transcription and ultimately apoptosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors. Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4's actions appear to be dependent on PGRMC1's function within the mitotic spindle.

Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytes at all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stages of the estrous cycle.

PGRMC1 (progesterone receptor membrane component 1) is part of a multi-protein complex, that is highly expressed in several cancers and is involved in chemoresistance. Although PGRMC1 plays an important role in various cancers, little is known about how PGRMC1 expression is regulated. Therefore, the present study was designed to elucidate the molecular mechanisms that influence PGRMC1 expression in ovarian cancer cells. An in silico approach revealed that the 3'-untranslated region of PGRMC1 contains one highly and one poorly conserved binding site for the microRNA let-7/miR-98 and one highly conserved binding site for miR-141/200a. Luciferase assays and real-time PCRs showed that the let-7 isoforms let-7i and miR-98 target PGRMC1 in SKOV-3 cells. In contrast, the conserved binding site for miR-200a/141 in the 3'-UTR of PGRMC1 is not functional. Stimulation of SKOV-3 cells with progesterone resulted in a decrease in PGRMC1 mRNA levels. Further, an analysis of endogenous let-7i levels in SKOV-3 cells revealed that let-7i expression increased after stimulation with progesterone. Therefore, progesterone may exert its effect on PGRMC1 expression in part by stimulation of let-7i. In conclusion, we propose that PGRMC1 expression is regulated by the miRNAs let-7/miR-98, which could become therapeutic targets, as PGRMC1, like many other targets of let-7, seems to be involved in cancer proliferation and chemotherapy resistance.

Although the mRNA that encodes progesterone receptor membrane component 1 (PGRMC1) is present in mammalian oocytes, nothing is known about either PGRMC1's expression pattern or function in oocytes during maturation, fertilization, and subsequent embryonic development. As PGRMC1 associates with the mitotic spindle in somatic cells, we hypothesized that PGRMC1 is involved in oocyte maturation (meiosis). Western blot analysis confirmed the presence of PGRMC1 in bovine oocytes. This study also shows that PGRMC1 is present at the germinal vesicle (GV)- and MII-stage oocytes and is associated with male and female pronucleus formation of the zygote and is highly expressed in blastocysts. A more detailed examination of PGRMC1 localization using confocal imaging demonstrated that in GV-stage oocytes, PGRMC1 was concentrated throughout the GV but did not localize to the chromatin. With the resumption of meiosis in vitro, PGRMC1 concentrated in the centromeric region of metaphase I chromosomes, while in the anaphase I/telophase I stages the majority of PGRMC1 concentrated between the separating chromosomes. At the metaphase II stage, PGRMC1 re-associated with the centromeric region of the chromosomes. A colocalization study demonstrated that PGRMC1 associated with the phosphorylated form of aurora kinase B, which localizes to the centromeres at metaphase. Finally, PGRMC1 antibody injection significantly lowered the percentage of oocytes that matured and reached the metaphase II stage after 24 h of culture. The majority of the PGRMC1 antibody-injected oocytes arrested in the prometaphase I stage of meiosis. Furthermore, in most of the PGRMC1 antibody-injected oocytes, the chromosomes were disorganized and scattered. Taken together, these data demonstrate that PGRMC1 is expressed in bovine oocytes and its localization changes at specific stages of oocyte maturation. These observations suggest an important role for PGRMC1 in oocyte maturation, which may be specifically related to the mechanism by which chromosomes segregate.

Progesterone receptor membrane component-1 (PGRMC1) is present in both the cytoplasm and nucleus of spontaneously immortalized granulosa cells (SIGCs). PGRMC1 is detected as a monomer in the cytoplasm and a DTT-resistant PGRMC1 dimer in the nucleus. Transfected PGRMC1-GFP localizes mainly to the cytoplasm and does not form a DTT-resistant dimer. Moreover, forced expression of PGRMC1-GFP increases the sensitivity of the SIGCs to progesterone (P4)'s anti-apoptotic action, indicating that the PGRMC1 monomer is functional. However, when endogenous PGRMC1 is depleted by siRNA treatment and replaced with PGRMC1-GFP, P4 responsiveness is not enhanced, although overall levels of PGRMC1 are increased. P4's anti-apoptotic action is also attenuated by actinomycin D, an inhibitor of RNA synthesis, and P4 activation of PGRMC1 suppresses Bad and increases Bcl2a1d expression. Taken together, the present studies suggest a genomic component to PGRMC1's anti-apoptotic mechanism of action, which requires the presence of the PGRMC1 dimer., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

IL-5 is a pleiotropic cytokine that promotes eosinophil differentiation and survival. While naïve bronchial epithelial cells (BEC) produce low levels of IL-5, the role of BEC-derived IL-5 in allergic airway inflammation is unknown. We now show that BEC, isolated from mice with OVA-induced allergic airway disease (AAD), produced elevated levels of IL-5 mRNA and protein as compared to BEC from naïve mice. To determine the contribution of BEC-derived IL-5 to effector responses in the airways, IL-5 deficient bone marrow chimeric mice were generated in which IL-5 expression was restricted to stromal (e.g. BEC) or hematopoietic cells. When subjected to AAD, IL-5 produced by BECs contributed to mucous metaplasia, airway eosinophilia, and OVA-specific IgA levels. Thus, IL-5 production by BEC can impact the microenvironment of the lung, modifying pathologic and protective immune responses in the airways., (2010 Elsevier Inc. All rights reserved.)

To determine whether progesterone receptor membrane component 1 (PGRMC1) regulates the development and cisplatin (CDDP)-sensitivity of human ovarian tumors, PGRMC1 was depleted from a human ovarian cancer cell line, dsRed-SKOV-3 cells, using a short hairpin RNA knockdown approach. Compared with parental dsRed-SKOV-3 cells, the PGRMC1-deplete cells grew slower in vitro and did not show progesterone's (P4) antiapoptotic effect. In fact, P4 induced apoptosis in PGRMC1-deplete cells in a dose-dependent manner. When transplanted into the peritoneum of athymic nude mice, parental dsRed-SKOV-3 cells developed numerous tumors, which were classified as either typical or oxyphilic clear cell tumors. CDDP increased the percentage of apoptotic nuclei in typical clear cell tumors and P4 attenuated CDDP-induced apoptosis. In contrast, the percentage of apoptotic nuclei in oxyphilic clear cell tumors was low (< or =1%) and was not significantly affected by CDDP and/or P4. Compared with tumors derived from parental dsRed SKOV-3 cells, PGRMC1-deplete tumors: 1) developed in fewer mice, 2) formed less frequently, 3) appeared smaller, and 4) resulted in fewer oxyphilic clear cell tumors. These PGRMC1-deplete tumors were not responsive to CDDP's apoptotic effects. The failure to respond to CDDP could be due to their poorly developed microvasculature system as judged by percentage of CD31-stained endothelial cells and/or their increased expression of ATP-binding cassette transporters, which are involved in drug resistance. Taken together, these findings indicate that PGRMC1 plays an essential role in the development and CDDP sensitivity of human ovarian tumors.

Context: Progesterone (P4) promotes its own secretion and the survival of human granulosa/luteal (GL) cells., Objective: The objective of these studies was to determine whether progesterone receptor membrane component-1 (PGRMC1) mediates P4's actions., Design: In vitro studies were conducted on GL cells from women undergoing in vitro fertilization and GL5 cells, which are derived from GL cells., Setting and Patients: GL cells were obtained from women undergoing fertility treatment at a university-based clinic and used for in vitro studies., Main Outcome Measures: PCR, Western blot, and immunocytochemistry were used to detect various progestin binding proteins. (3)H-P4 binding kinetics were assessed on partially purified PGRMC1. Apoptosis was determined after culture by either TUNEL or DAPI staining. P4 was measured by an ELISA assay. PGRMC1 was depleted using small interfering RNA., Results: GL and GL5 cells expressed several P4 binding proteins including the nuclear progesterone receptor (PGR), progestin/adipoQ receptors (PAQR 7, 8, and 5) and PGRMC1. Ligand binding studies revealed that both P4 and the progestin, R5020, bound PGRMC1 with an EC(50) of approximately 10 nm. Interestingly, P4 inhibited apoptosis at concentrations in the 10 nm range, whereas R5020 stimulated P4 secretion at concentrations of at lease 16 mum. Depleting PGRMC1 attenuated P4's antiapoptotic action but failed to influence R5020-induced P4 secretion., Conclusions: These studies conclusively demonstrate that in human GL cells PGRMC1 functions as a receptor through which P4 activates a signal cascade that prevents apoptosis. In contrast, PGRMC1 does not mediate P4's ability to acutely promote its own secretion.

The protein now called Progesterone Receptor Membrane Component 1 (PGRMC1) has been described independently by many groups in different cellular contexts. As a result it has been given an impressive diversity of names. While this protein was initially described on the basis of a singular property, e.g. expression or steroid binding, its possible physiological roles have only recently been reported. Current evidence supports the perception that PGRMC1 may be involved in sterol metabolism or homeostasis and cell survival. Here, we summarize a few sometimes neglected pieces of evidence from the literature along with unpublished findings on the properties and functions of PGRMC1.

Although activation of the nuclear progesterone (P(4)) receptor (PGR) is required for uterine function, some of the actions of P(4) are mediated through a PGR-independent mechanism. The receptors that account for the PGR-independent actions have not been identified with certainty. The purpose of this study was to assess the expression, localization and hormonal regulation of two novel P(4) receptor candidates, P(4) receptor membrane component (PGRMC) 1 and PGRMC2, as well as the PGRMC1 partner Serpine 1 mRNA binding protein (SERBP1). Unlike Pgrmc1 and Serbp1, which remained unchanged throughout the estrous cycle, Pgrmc2 was highly up-regulated during proestrus and metestrus. Immunohistochemical analyses suggest that PGRMC1 and SERBP1 promote differentiation, since the expression of these proteins increased in endometrial cells undergoing steroid-depended terminal differentiation. Progesterone rather than estrogen appears to be primarily responsible for up-regulating the expression of PGRMCs. PGRMC1 and SERBP1 also showed overlapping patterns of expression in the human placenta and associated membranes with the most abundant expression in smooth muscle of the placental vasculature, villous capillaries and the syncytiotrophoblast. Based on these findings, it is proposed that PGRMC1:SERBP1 protein complex functions in events important to early pregnancy including cellular differentiation, modulation of apoptosis and steroidogenesis. These studies provide a platform from which to build a clearer understanding of P(4) actions in the female reproductive tract and placental tissues that are mediated by non-classical mechanisms.

Context: Progesterone (P4) influences ovarian cancer cells by an unknown mechanism., Objective: The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers., Design, Setting and Patients: Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells., Main Outcome Measures: PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1's role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1's action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining., Results: PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP., Conclusions: These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1's action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.

We have followed throughout time the development of allergic airway disease (AAD) in both uninfected mice and mice infected intranasally with murine cytomegalovirus (MCMV). Histological evaluation of lung tissue from uninfected mice with AAD demonstrated mucus plugging after 14 and 21 days of ovalbumin-aerosol challenge, with resolution of mucus plugging occurring by 42 days. In MCMV/AAD mice, mucus plugging was observed after 7 days of ovalbumin-aerosol challenge and remained present at 42 days. The level of interleukin-13 in bronchoalveolar lavage fluid from MCMV/AAD mice was decreased compared with AAD mice and was accompanied by increased levels of interferon-gamma. Levels of Muc5A/C, Muc5B, or Muc2 mucin mRNA in the lungs of MCMV/AAD mice were not elevated compared with AAD mice. MCMV was able to infect the airway epithelium, resulting in decreased expression of Foxj1, a transcription factor critical for ciliogenesis, and a loss of ciliated epithelial cells. In addition, an increase in the number of epithelial cells staining positive for periodic acid-Schiff was observed in MCMV/AAD airways. Together, these findings suggest that MCMV infection of the airway epithelium enhances goblet cell metaplasia and diminishes efficient mucociliary clearance in mice with AAD, resulting in increased mucus plugging.

Progesterone (P4) receptor membrane component-1 (PGRMC1) and its binding partner, plasminogen activator inhibitor 1 RNA binding protein (PAIRBP1) are thought to form a complex that functions as membrane receptor for P4. The present investigations confirm PGRMC1's role in this membrane receptor complex by demonstrating that depleting PGMRC1 with PGRMC1 small interfering RNA results in a 60% decline in [(3)H]P4 binding and the loss of P4's antiapoptotic action. Studies conducted on partially purified GFP-PGRMC1 fusion protein indicate that [(3)H]P4 specifically binds to PGRMC1 at a single site with an apparent K(d) of about 35 nm. In addition, experiments using various deletion mutations reveal that the entire PGRMC1 molecule is required for maximal [(3)H]P4 binding and P4 responsiveness. Analysis of the binding data also suggests that the P4 binding site is within a segment of PGRMC1 that is composed of the transmembrane domain and the initial segment of the C terminus. Interestingly, PAIRBP1 appears to bind to the C terminus between amino acids 70-130, which is distal to the putative P4 binding site. Taken together, these data provide compelling evidence that PGRMC1 is the P4 binding protein that mediates P4's antiapoptotic action. Moreover, the deletion mutation studies indicate that each domain of PGRMC1 plays an essential role in modulating PGRMC1's capacity to both bind and respond to P4. Additional studies are required to more precisely delineate the role of each PGRMC1 domain in transducing P4's antiapoptotic action.

This review summarizes recent findings on the non-genomic or membrane-initiated actions of progesterone that regulate the function of the normal and neoplastic mammalian ovary. This review focuses on three receptors: the classic progesterone receptor, the membrane progesterone receptors (MPRalpha, beta, and gamma) that were initially cloned from seatrout ovaries, and a progesterone binding protein referred to as progesterone receptor membrane component-1 (PGRMC1). Specifically, the structure of each of these receptors is compared and related to their capacity to activate various signal transduction pathways. Then the biological effects of P4 on the function of granulosa cells, luteal cells, ovarian surface epithelial cells, and ovarian cancers that are derived from the ovarian surface epithelial cells are discussed in relationship to the expression of each of these receptors. Whenever possible, studies involving human cells and tissues are presented, although data from other mammalian species are used to supplement the human studies to provide a more complete picture of this complex and rapidly developing area of membrane-initiated actions of progesterone.

The present studies were designed to 1) describe changes in both the mitochondrial membrane potential and ATP content of spontaneously immortalized granulosa cells as they undergo apoptosis, 2) identify some of the downstream events that are activated by progesterone (P4), and 3) relate these downstream events to changes in mitochondrial function and apoptotic cell death. These studies revealed that in response to serum deprivation, the mitochondrial membrane potential initially hyperpolarizes and ATP content increases. That this increase in ATP is required for apoptosis was demonstrated by the finding that oligomycin inhibited the increase in ATP and apoptosis. Piridoxalphosphate-6-azopeyl-2'-4'-disulfonic acid, an inhibitor of purinergic receptors, which are activated by ATP, also inhibited apoptosis due to serum withdrawal. This study provides additional support for ATP's causative role in apoptosis. Moreover, 8-Br-cGMP, a protein kinase G (PKG) activator, mimicked P4's action, whereas a PKG antagonist, DT-3, attenuated P4's suppressive effect on ATP and apoptosis. Finally, DT-3 treatment was shown to attenuate P4-regulated phosphorylation of 14-3-3sigma and its binding partner, ATP synthasebeta/precursor and the amount of ATP synthasebeta/precursor that bound to 14-3-3sigma. Based on these data, it is proposed that P4 prevents apoptosis in part by activating PKG, which in turn maintains the interaction between ATP synthasebeta/precursor and 14-3-3sigma. In the absence of P4-induced PKG activity, we further propose that some ATP synthasebeta precursor dissociates from 14-3-3sigma, resulting in its activation and incorporation into the ATP synthase complex, which ultimately results in an increase in ATP and apoptosis.

Context: Progesterone (P4) inhibits human granulosa/luteal cell apoptosis by an unknown mechanism., Objective: Our objective was to assess the role of the nuclear P4 receptor (PGR) and PGR membrane component 1 (PGRMC1) in mediating P4's antiapoptotic action in human granulosa/luteal cells., Design, Setting, and Patients: In vitro laboratory studies were designed in which human granulosa/luteal cells were harvested from in vitro fertilization patients from 2004-2006., Main Outcome Measure: Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays and DNA staining. Protein expression was observed by Western blot and immunocytochemistry., Results: PGR was detected in 20% of the human granulosa/luteal cells, and 25 and 50 microM RU486 induced at least 70% of the cells to undergo apoptosis. Five micromolar RU486 neither induced apoptosis nor attenuated the antiapoptotic action of 1 microM P4. PGRMC1 and its binding partner, plasminogen activator inhibitor RNA-binding protein-1 (PAIRBP1), were detected in human granulosa/luteal cells. Antibodies to either PGRMC1 or PAIRBP1 completely attenuated P4's action., Conclusions: PGR does not exclusively mediate P4's action because 1) 5 microM RU486 should have been able to override the antiapoptotic action of 1 microM P4 because RU486 binds to the PGR at a greater affinity than P4; 2) 25 and 50 microM RU486 induce three to four times more cells to undergo apoptosis than express PGR; 3) P4 must be continuously present to prevent apoptosis, which implies a rapid, possibly membrane-initiated mechanism of action; and 4) expression and blocking antibody studies suggest that PGRMC1 and PAIRBP1 account in part for P4's action in human granulosa/luteal cells.

This minireview summarizes the role that progesterone (P4) plays in regulating granulosa and luteal cell function. These actions include the stimulation of P4 synthesis and the inhibition of estrogen synthesis, mitosis, and apoptosis. P4 also plays a key role in the ovulatory process. Although P4's actions are well documented, the mechanism or mechanisms that mediate all of these actions have not been defined. In addition to P4-induced gene transcription that is mediated by the nuclear P4 receptors (PGR-A and PGR-B), three other receptor/signal transduction pathways could account for P4's intraovarian actions. These pathways could be mediated by 1) the PGR localizing at or near the plasma membrane and activating SRC family kinases, 2) a membrane progestin receptor that responds to P4 by lowering intracellular cAMP and increasing MAPK 3/1 activity, and 3) a membrane receptor complex composed of serpine 1 mRNA binding protein (also known as PAIRBP1 or RDA288) and progesterone receptor membrane component 1. Ligand activation of this complex likely leads to an increase in protein kinase G activity, the maintenance of low basal intracellular free calcium, and the inhibition of granulosa and luteal cell mitosis and apoptosis. Given the complexity of P4's actions within the ovary, it is likely that all of these receptor/signal transduction pathways influence some aspect of ovarian function with the specific P4 response dependent on 1) the expression pattern of these putative P4 receptors, 2) the P4 binding affinity of each receptor system, and 3) the amount of available P4.

Progesterone receptor membrane component-1 (PGRMC1) interacts with plasminogen activator inhibitor RNA binding protein-1 (PAIRBP1), a membrane-associated protein involved in the antiapoptotic action of progesterone (P4). In this paper, the first studies were designed to assess the ovarian expression pattern of PGRMC1 and PAIRBP1. Western blot analysis revealed that spontaneously immortalized granulosa cells (SIGCs) as well as granulosa and luteal cells express both proteins. Luteal cells were shown to express more PGRMC1 than granulosa cells. Immunohistochemical studies confirmed this and demonstrated that PGRMC1 was present in thecal/stromal cells, ovarian surface epithelial cells, and oocytes. PAIRBP1 was also expressed in thecal/stromal cells and ovarian surface epithelial cells but not oocytes. Furthermore, PAIRBP1 and PGRMC1 were detected among the biotinylated surface proteins that were isolated by avidin affinity purification, indicating that they localized to the extracellular surface of the plasma membrane. Confocal microscopy revealed that both of these proteins colocalize to the plasma membrane as well as the cytoplasm. The second studies were designed to assess PGRMC1's role in P4's antiapoptotic actions. These studies showed that overexpression of PGRMC1 increased 3H-P4 binding and P4 responsiveness. Conversely, treatment with a PGRMC1 antibody blocked P4's antiapoptotic action. Taken together, the present findings indicate that both PAIRBP1 and PGRMC1 show a similar expression pattern within the ovary and colocalize to the extracellular surface of the plasma membrane. At the plasma membrane, these two proteins interact to form a complex that is required for P4 to transduce its antiapoptotic action.

The effect of granulosa cell contact on the ability of zona-free oocytes to undergo germinal vesicle breakdown (GVBD) was assessed using a granulosa cell co-culture system. Oocytes contacted granulosa cells in a site-specific manner such that their GV was away from the granulosa cells. Also contact with granulosa cells reduced the percentage of oocytes undergoing GVBD from about 40% to 15%. GVBD was inhibited by contact with granulosa cells but not a granulosa cell-secreted product, since oocytes cultured in the same culture, that were adjacent to the granulosa cell monolayer underwent GVBD at the same rate as controls. Similarly, media collected from granulosa cell cultures did not attenuate the rate of GVBD. The ability of granulosa cell contact to inhibit GVBD was equal to that of db-cAMP. Moreover, the ability of granulosa cells to inhibit GVBD was not mimicked by spontaneously immortalized granulosa cells. This cell specificity appeared to be related to N-cadherin, since granulosa cells and oocytes express N-cadherin and a N-cadherin antibody attenuates the effect of granulosa cell contact. The mechanism through which N-cadherin mediated cell contact maintains meiotic arrest is unknown. It is possible that homophilic N-cadherin binding between the granulosa cells and oocyte acts through a junxtacrine mechanism, which in part may lead in the activation fibroblast growth factor (FGF) receptors that are expressed by the oocyte. The involvement of FGF receptors is supported by the observations that FGF and a N-cadherin peptide known to activate FGF receptors inhibit GVBD.

The protein PAIRBP1, which was initially referred to as RDA288, is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole-cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal, and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast, eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, nonpermeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4's antiapoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4's action, it did not reduce the capacity of cells to specifically bind (3)H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as progesterone receptor membrane complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4's antiapoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1's action remains to be elucidated, but it may involve an interaction with PGRMC1.

Progesterone (P4) inhibits granulosa cell and spontaneously immortalized granulosa cell (SIGC) apoptosis by regulating membrane-initiated events. However, the nature of the signal transduction pathway that is induced by these membrane-initiated events has not been defined. To gain insights into the P4-regulated signal transduction pathway, mouse granulosa cells and SIGCs were cultured with 8-br-cGMP and P4. In culture, 8-br-cGMP mimicked P4's antiapoptotic actions. Because cGMP activates protein kinase G (PKG), the effect of PKG antagonists on P4-regulated SIGC viability was assessed. P4's antiapoptotic action was attenuated by the PKG inhibitors, Rp-8-pCPT-cGMP, KT5823, the PKG-1alpha-specific inhibitor, DT-3, and a dominant negative PKG-1alpha. Further, the type I isoform of PKG was shown to be expressed by SIGCs and activated by P4. P4's antiapoptotic action was not affected by the PKA inhibitor, KT5720. Collectively, these findings indicate that P4 maintains SIGC viability by activating PKG-1alpha. PKG-1alpha-GFP was shown to localize predominantly to the cytoplasm of SIGCs. To identify potential cytoplasmic targets of PKG-1alpha, SIGCs were cultured for 5 h with P4 in the presence or absence of DT-3. Cell lysates were prepared and subjected to two-dimensional electrophoresis. The resulting gels were sequentially stained with ProQ-Diamond Gel Stain and Coomassie Blue to reveal phosphorylated proteins. The two-dimensional gels revealed one major protein, the phosphorylation status of which was abrogated by DT-3. Mass spectrometric analysis identified this protein as 14-3-3sigma, with 14-3-3sigma being phosphorylated on tyrosine 19, serine 28, serine 69, serine 74, threonine 90, threonine 98, and serine 116. Finally, difopein, a specific 14-3-3 inhibitor, was shown to induce apoptosis even in the presence of serum. These data suggest that 1) P4 regulates the phosphorylation status of 14-3-3sigma through a PKG-dependent pathway and 2) 14-3-3sigma plays a central and essential role in maintaining the viability of SIGCs.