Cyclic Regulation of Sensory Perception by a Female Hormone Alters Behavior.

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.

PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. 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 unknown3,4. 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 defined3,4. 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.

Supplementing culture medium with the weak acid, 5,5-dimethyl-2,4-oxazolidinedione (DMO) limits the development of aneuploid mouse embryos through a Trp53-dependent mechanism.

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.

Necessity is the mother of invention and the evolutionary force driving the success of in vitro fertilization.

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.

MicroRNA-21 as a regulator of human cumulus cell viability and its potential influence on the developmental potential of the oocyte.

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.

Progesterone receptor membrane component 1 and 2 regulate granulosa cell mitosis and survival through a NFΚB-dependent mechanism†.

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.

PGRMC1/2 promotes luteal vascularization and maintains the primordial follicles of mice

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.

PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes.

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 (H2O2) 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.

AG 205, a progesterone receptor membrane component 1 antagonist, ablates progesterone's ability to block oxidative stress-induced apoptosis of human granulosa/luteal cells†.

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.

Progestin and AdipoQ Receptor 7, Progesterone Membrane Receptor Component 1 (PGRMC1), and PGRMC2 and Their Role in Regulating Progesterone's Ability to Suppress Human Granulosa/Luteal Cells from Entering into the Cell Cycle.

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.

Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells.

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.

Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

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.

Non-canonical progesterone signaling in granulosa cell function.

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.

Plasminogen activator inhibitor 1 RNA-binding protein interacts with progesterone receptor membrane component 1 to regulate progesterone's ability to maintain the viability of spontaneously immortalized granulosa cells and rat granulosa cells.

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.

Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B.

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.

Nonerythroid hemoglobin promotes human cumulus cell viability and the developmental capacity of the human oocyte.

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% O2. 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% O2 vs. 20% O2, respectively, and multiple regulators of oxidative stress were overexpressed in cells cultured under 20% O2 compared with those under 5% O2. However, the rate of apoptosis and amount of mitochondrial reactive oxidative species increased by 1.25-fold in CCs cultured under 20% O2 compared with those under 5% O2. 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.

Progesterone Signaling and Mammalian Ovarian Follicle Growth Mediated by Progesterone Receptor Membrane Component Family Members.

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.

A novel role for progesterone and progesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis.

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.

Progesterone Receptor Membrane Component (PGRMC)1 and PGRMC2 and Their Roles in Ovarian and Endometrial Cancer.

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.

Bronchial epithelial cells produce IL-5: implications for local immune responses in the airways.

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.