P38α MAPK is a gatekeeper of uterine progesterone responsiveness at peri-implantation via Ube3c-mediated PGR degradation.

Estrogen and progesterone specify the establishment of uterine receptivity mainly through their respective nuclear receptors, ER and PR. PR is transcriptionally induced by estrogen-ER signaling in the endometrium, but how the protein homeostasis of PR in the endometrium is regulated remains elusive. Here, we demonstrated that the uterine-selective depletion of P38α derails normal uterine receptivity ascribed to the dramatic down-regulation of PR protein and disordered progesterone responsiveness in the uterine stromal compartment, leading to defective implantation and female infertility. Specifically, Ube3c, an HECT family E3 ubiquitin ligase, targets PR for polyubiquitination and thus proteasome degradation in the absence of P38α. Moreover, we discovered that P38α restrains the polyubiquitination activity of Ube3c toward PR by phosphorylating the Ube3c at serine741 . In summary, we provided genetic evidence for the regulation of PR protein stability in the endometrium by P38α and identified Ube3c, whose activity was modulated by P38α-mediated phosphorylation, as an E3 ubiquitin ligase for PR in the uterus.

ACE2: a key modulator of the renin-angiotensin system and pregnancy.

Angiotensin-converting enzyme 2 (ACE2) is a membrane-bound protein containing 805 amino acids. ACE2 shows approximately 42% sequence similarity to somatic ACE but has different biochemical activities. The key role of ACE2 is to catalyze the vasoconstrictor peptide angiotensin (ANG) II to Ang-(1-7), thus regulating the two major counterbalancing pathways of the renin-angiotensin system (RAS). In this way, ACE2 plays a protective role in end-organ damage by protecting tissues from the proinflammatory actions of ANG II. The circulating RAS is activated in normal pregnancy and is essential for maintaining fluid and electrolyte homeostasis and blood pressure. Renin-angiotensin systems are also found in the conceptus. In this review, we summarize the current knowledge on the regulation and function of circulating and uteroplacental ACE2 in uncomplicated and complicated pregnancies, including those affected by preeclampsia and fetal growth restriction. Since ACE2 is the receptor for SARS-CoV-2, and COVID-19 in pregnancy is associated with more severe disease and increased risk of abnormal pregnancy outcomes, we also discuss the role of ACE2 in mediating some of these adverse consequences. We propose that dysregulation of ACE2 plays a critical role in the development of preeclampsia, fetal growth restriction, and COVID-19-associated pregnancy pathologies and suggest that human recombinant soluble ACE2 could be a novel therapeutic to treat and/or prevent these pregnancy complications.

Characterization of Glutathione Peroxidase 4 in Rat Oocytes, Preimplantation Embryos, and Selected Maternal Tissues during Early Development and Implantation.

This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 µL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

The histone methyltransferase EZH2 is required for normal uterine development and function in mice†.

Enhancer of zeste homolog 2 (EZH2) is a rate-limiting catalytic subunit of a histone methyltransferase, polycomb repressive complex, which silences gene activity through the repressive histone mark H3K27me3. EZH2 is critical for epigenetic effects of early estrogen treatment, and may be involved in uterine development and pathologies. We investigated EZH2 expression, regulation, and its role in uterine development/function. Uterine epithelial EZH2 expression was associated with proliferation and was high neonatally then declined by weaning. Pre-weaning uterine EZH2 expression was comparable in wild-type and estrogen receptor 1 knockout mice, showing neonatal EZH2 expression is ESR1 independent. Epithelial EZH2 was upregulated by 17ß-estradiol (E2) and inhibited by progesterone in adult uteri from ovariectomized mice. To investigate the uterine role of EZH2, we developed a EZH2 conditional knockout (Ezh2cKO) mouse using a cre recombinase driven by the progesterone receptor (Pgr) promoter that produced Ezh2cKO mice lacking EZH2 in Pgr-expressing tissues (e.g. uterus, mammary glands). In Ezh2cKO uteri, EZH2 was deleted neonatally. These uteri had reduced H3K27me3, were larger than WT, and showed adult cystic endometrial hyperplasia. Ovary-independent uterine epithelial proliferation and increased numbers of highly proliferative uterine glands were seen in adult Ezh2cKO mice. Female Ezh2cKO mice were initially subfertile, and then became infertile by 9 months. Mammary gland development in Ezh2cKO mice was inhibited. In summary, uterine EZH2 expression is developmentally and hormonally regulated, and its loss causes aberrant uterine epithelial proliferation, uterine hypertrophy, and cystic endometrial hyperplasia, indicating a critical role in uterine development and function.

Can the negative effects of ketamine abuse on female genital organs be prevented by nimesulide? An experimental study.

The objective of this study is to investigate the effects of nimesulide on ketamine-induced ovarian and uterine toxicity by biochemical and histopathological examinations. Ketamine is an anesthetic agent whose use leads to overproduction of catecholamines. Nimesulide is a cyclooxygenase-2 inhibitor, which has also been reported to exert a significant antioxidant effect. Wistar albino female rats were randomly divided into three groups as follows: ketamine group (60 mg/kg), ketamine (60 mg/kg) + nimesulide (50 mg/kg) group, and a healthy control group. Then, the biochemical levels and histopathological findings in the ovaries and uteri of the rats were examined for malondialdehyde, myeloperoxidase, total glutathione and superoxide dismutase. The study demonstrated that, in the uterine and ovarian tissues of rats that have been administered ketamine, there was a decrease in the levels of total glutathione and superoxide dismutase, while malondialdehyde and myeloperoxidase was increased: however it was observed that these ratios were reversed in the ketamine+nimesulide group. It was also proved that the negative effects of ketamine can be corrected with nimesulide when the myometrial and endometrial thicknesses are compared. Antioxidants such as nimesulide may protect against the damage caused by ketamine to the genital organs in young women.

Endocannabinoids as endometrial inflammatory markers in lactating Holstein cows.

The objective of this study was to consider endocannabinoid system as inflammatory markers in bovine endometrium to better understand the role of this system in regulating many of the functions that are related to inflammatory condition. At day 26 post-partum, fourteen cows were divided into two groups depending on the inflammatory condition: 1- subclinical endometritis (n = 7, with purulent or mucopurulent uterine discharge detectable in the vagina) and 2- healthy (n = 7, No (muco)) purulent discharge. Blood samples were collected at 26 and 30 days relative to calving to determine plasma tumour necrosis factor (TNF) and lipopolysaccharide-binding protein (LBP) concentrations; moreover, uterine biopsy was carried out on day 26 post-partum to measure mRNA abundance of TNF, interleukin-1B (IL1B), interleukin-6 (IL-6), C-X-C motif chemokine ligand 8 (CXCL8), endocannabinoid receptor (CNR2), N-acyl phosphatidylethanolamine phospholipase D (NAPEPLD), fatty acid amide hydrolase (FAAH), N-acylethanolamine acid amidase (NAAA) and monoglyceride lipase (MGLL) by real-time PCR. Results showed mean plasma concentrations of TNF and LBP were lower in healthy cows compared to subclinical endometritis cows (p < .05). Relative mRNA expression for NAAA and FAAH was decreased (p < .05), and relative mRNA expression for CNR2 and NAPEPLD increased in cows with subclinical endometritis compared to healthy cows. In conclusion, relative mRNA expression of TNF, IL1B and CXCL8 and plasma concentration of LBP increased during inflammatory condition along with decreased endocannabinoids hydrolyzing enzyme (NAAA and FAAH), increased enzymes that synthesize endocannabinoids (NAPEPLD) and relative gene expression of the endocannabinoid receptor; together, these contribute to increased endocannabinoids levels during inflammation. Overall, we provide evidence that endocannabinoid system is altered in endometrium tissue during inflammation through increased mRNA expression of CNR2 and synthesis enzyme and decreased mRNA expression of hydrolyzing enzymes interfere with pro-cytokine production and signalling, which may interfere with the onset and progression of inflammation.

Immunohistochemical Features of O6-Methylguanine-DNA Methyltransferase Expression during Ovarian Endometriosis.

A comparative immunohistochemical study for the expression of O6-methylguanine-DNA methyltransferase (MGMT) was performed in tissues of the eutopic endometrium and ovarian endometriosis. The highest level of MGMT expression in eutopic endometrial tissue was observed in epitheliocyte nuclei during the proliferative phase. In regions of endometriosis the expression of MGMT in epitheliocyte nuclei was shown to increase during stages I and II, but decreased in stages III and IV. The progression of endometriosis was accompanied by a gradual increase of study parameters in the nuclei and cytoplasm of stromal cells. These changes reflect the impairment of DNA reparation, which probably serves as a stage in the development and progression of endometriosis.

Increased vascular and uteroplacental matrix metalloproteinase-1 and -7 levels and collagen type I deposition in hypertension in pregnancy: role of TNF-α.

Preeclampsia is a pregnancy-related disorder manifested as maternal hypertension in pregnancy (HTN-Preg) and fetal growth restriction. Placental ischemia could be an initiating event that leads to abnormal vascular and uteroplacental remodeling in HTN-Preg; however, the molecular targets and intermediary mechanisms involved are unclear. We tested the hypothesis that placental ischemia could target vascular and uteroplacental matrix metalloproteinases (MMPs) through an inflammatory cytokine-mediated mechanism. MMP levels and distribution were measured in the aorta, uterus, and placenta of normal pregnant (Preg) rats and pregnant rats with reduced uterine perfusion pressure (RUPP). Maternal blood pressure was higher and the litter size and pup weight were lower in RUPP compared with Preg rats. Gelatin zymography showed prominent uterine MMP-2 and MMP-9 activity that was dependent on the amount of loaded protein. At saturating protein loading, both gelatin and casein zymography revealed two additional bands corresponding to MMP-1 and MMP-7 that were greater in the aorta, uterus, and placenta of RUPP compared with Preg rats. Western blots and immunohistochemistry confirmed increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of RUPP versus Preg rats. The levels of MMP-1 and MMP-7 substrate collagen type I were greater in tissues of RUPP compared with Preg rats. In organ culture, TNF-α increased MMP-1 and MMP-7 in the aorta, uterus, and placenta of Preg rats, and a TNF-α antagonist prevented the increases in MMPs in tissues of RUPP rats. Thus, placental ischemia, possibly through TNF-α, increases vascular and uteroplacental MMP-1 and MMP-7, which, in turn, alter collagen deposition and cause inadequate tissue remodeling in HTN-Preg. Cytokine antagonists may reverse the increase in MMP-1 and MMP-7 expression/activity and, in turn, restore proper vascular and uteroplacental remodeling in HTN-Preg and preeclampsia.NEW & NOTEWORTHY The molecular mechanisms of preeclampsia are unclear, making it difficult to predict, prevent, or manage the pregnancy-associated disorder. This study showed that placental ischemia, possibly through the release of TNF-α, causes increases in the levels of matrix metalloproteinase (MMP)-1 and MMP-7, which could alter collagen deposition and cause inadequate uteroplacental and vascular remodeling in hypertension in pregnancy. The data suggest that targeting MMP-1 and MMP-7 and their upstream modulators, such as TNF-α, could provide a new approach in the management of hypertension in pregnancy and preeclampsia.

Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation.

Successful implantation requires intimate interactions between a competent blastocyst and a receptive uterus. We recently demonstrated that the aberrant activation of opioid signaling by exogenous ligands adversely affects preimplantation embryonic development and subsequent implantation in mice. However, the underlying machinery governing the dynamic homeostasis of the endogenous opioid system in the uterus during early pregnancy remains elusive. We now show that all three major endogenous opioid precursors are spatiotemporally expressed in the uterus during early pregnancy. Moreover, we observe the well-coordinated expression of the synthetic enzyme prohormone convertases 1/3 (PC1/3) at lower levels and of its inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor (Pcsk1n) and the degrading enzyme membrane metallo-endopeptidase (MME) at higher levels in the receptive uterus. Both estrogen and progestin tend to reduce the uterine levels of opioid ligand precursors in the ovariectomized mouse model. This tight regulation of the endogenous opioid system by PC1/3, Pcsk1n and MME has been further confirmed in physiologically related pseudopregnancy and delayed implantation mouse models. The coordinated regulation of opioid precursor biosynthesis and metabolism helps to create appropriate opioid signaling ensuring uterine receptivity for implantation. Thus, endogenous uterine opioid levels are primarily determined by the coordinated expressions of PC1/3, Pcsk1n and MME under the influence of ovarian progestin and estrogen. Our findings raise an additional cautionary note regarding the effects of opioid abuse on early pregnancy events.

l-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect.

Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l-cysteine (l-Cys) by cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H2S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert l-Cys into H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, l-Cys, sodium hydrogen sulfide or sildenafil but not d-Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE(-/-) mice. Indeed, the increase in H2S production mediated by l-Cys or by sildenafil was not found in CSE(-/-) mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE(-/-) mice. A decisive proof for the involvement of H2S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, l-Cys/CSE/H2S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders.

A high dose mode of action for tetrabromobisphenol A-induced uterine adenocarcinomas in Wistar Han rats: A critical evaluation of key events in an adverse outcome pathway framework.

TBBPA is a non-genotoxic flame retardant used to improve fire safety in a wide variety of consumer products. Estimated human exposures to TBBPA are very low (<0.000084 mg/kg-day), relative to the doses (500 and 1000 mg/kg-day of TBBPA) administered in a recent bioassay that resulted in uterine tumors in Wistar Han rats following chronic exposure. As part of an effort to characterize the relevance of the uterine tumors to humans, data and biological knowledge relevant to the progression of events associated with TBBPA-induced uterine tumors in female rats were organized in an adverse outcome pathway (AOP) framework. Based on a review of possible MOAs for chemically induced uterine tumors and available TBBPA data sets, a plausible molecular initiating event (MIE) was the ability of TBBPA to bind to and inhibit estrogen sulfotransferases, the enzymes responsible for sulfation of estradiol. Subsequent key events in the AOP, including increased bioavailability of unconjugated estrogens in uterine tissue, would occur as a result of decreased sulfation, leading to a disruption in estrogen homeostasis, increased expression of estrogen responsive genes, cell proliferation, and hyperplasia. Available data support subsequent key events, including generation of reactive quinones from the metabolism of estrogens, followed by DNA damage that could contribute to the development of uterine tumors. Uncertainties associated with human relevance are highlighted by potential strain/species sensitivities to development of uterine tumors, as well as the characterization of a dose-dependent MIE. For the latter, it was determined that the TBBPA metabolic profile is altered at high doses (such as those used in the cancer bioassay), and thus an MIE that is only operative under repeated high dose, administration. The MIE and subsequent key events for the development of TBBPA-induced uterine tumors are not feasible in humans given differences in the kinetic and dynamic factors associated with high dose exposures in rats relative to human exposure levels to TBBPA.

Distinct expression and localization of the type II diacylglycerol kinase isozymes δ, η and κ in the mouse reproductive organs.

BACKGROUND: We have revealed that the type II diacylglycerol kinases (DGKs) δ, η and κ were expressed in the testis and ovary. However, these enzymes' functions in the reproductive organs remain unknown. RESULTS: In this study, we first identified the expression sites of type II DGKs in the mouse reproductive organs in detail. Reverse transcription-polymerase chain reaction and Western blotting confirmed that DGKδ2 (splicing variant 2) but not DGKδ1 (splicing variant 1) and DGKκ were expressed in the testis, ovary and uterus. DGKη1 (splicing variant 1) but not DGKη2 (splicing variant 2) was strongly detected in the ovary and uterus. Interestingly, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the testis. Moreover, we investigated the distribution of type II DGKs in the testis, ovary and uterus through in situ hybridization. DGKδ2 was distributed in the primary spermatocytes of the testis and ovarian follicles. DGKη1 was distributed in the oviductal epithelium of the ovary and the luminal epithelium of the uterus. Intriguingly, DGKη3 was strongly expressed in the secondary spermatocytes and round spermatids of the testis. DGKκ was distributed in the primary and secondary spermatocyte of the testis. CONCLUSION: These results indicate that the expression patterns of the type II DGK isoforms δ2, η1, η3 and κ differ from each other, suggesting that these DGK isoforms play specific roles in distinct compartments and developmental stages of the reproductive organs, especially in the processes of spermatogenesis and oocyte maturation.

Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality.

Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain.

Apoptosis and cell proliferation in the mouse model of embryonic death induced by Tritrichomonas foetus infection.

Bovine tritrichomonosis is a sexually transmitted disease caused by the protozoon Tritrichomonas foetus and characterised by embryonic-death and abortion. During pregnancy, the processes of cell proliferation and death play a crucial role for blastocyst implantation and the subsequent maintenance of early pregnancy, and their misbalance may lead to the abortion. In this study, we aimed to investigate whether cell proliferation and death may be altered during tritrichomonosis. For this purpose, we used pregnant BALB/c mice as an alternative experimental animal model that has successfully reproduced the infection. We analysed the immunohistochemical expression of active caspase-3 and proliferating cell nuclear (PCNA) antigens in the endometrium of infected mice. We found an increase in the number of caspase-3 positive cells in infected mice that were not pregnant at the necropsy. Besides, the number of positive proliferating cells increased in the uterine luminal epithelium of infected animals killed at 5-7 days post coitum (dpc). Pregnant infected mice killed at 8-11 dpc showed higher proliferation than control animals. We suggest that the cytopathic effect induced by T. foetus in the uteri of infected mice may induce the apoptosis of the epithelial cells and, as a result, promote a compensatory proliferative response. The information described here will be helpful to further study the pathogenesis of the bovine tritrichomonosis.

Expression, activation, and role of AKT isoforms in the uterus.

The three isoforms of AKT: AKT1, AKT2, and AKT3, are crucial regulators of both normal and pathological cellular processes. Each of these isoforms exhibits a high level of homology and functional redundancy with each other. However, while being highly similar and structurally homologous, a rising amount of evidence is showing that each isoform possesses specific targets as well as preferential subcellular localization. The role of AKT has been studied extensively in reproductive processes, but isoform-specific roles are yet to be fully understood. This review will focus on the role of AKT in the uterus and its function in processes related to cell death and proliferation such as embryo implantation, decidualization, endometriosis, and endometrial cancer in an isoform-centric manner. In this review, we will cover the activation of AKT in various settings, localization of isoforms in subcellular compartments, and the effect of isoform expression on cellular processes. To fully understand the dynamic molecular processes taking place in the uterus, it is crucial that we better understand the physiological role of AKT isoforms as well as their function in the emergence of diseases.

Inhibitory effect of Nodal on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma of uterus.

Cancers consist of heterogeneous populations. Recently, it has been demonstrated that cells with tumorigenic potential are limited to a small population, called cancer-initiating cells (CICs). Aldehyde dehydrogenase 1 (ALDH1) is one of the markers of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and ALDH1-high population of endometrioid adenocarcinoma cell line was more tumorigenic, resistant to anti-cancer drugs, and invasive than ALDH1-low population. Here, the regulatory signaling for ALDH1 was examined. The inhibition of TGF-ß signaling increased ALDH1-high population. Among TGF-ß family members, Nodal expression and ALDH1 expression levels were mutually exclusive. Immunohistochemical analysis on clinical samples revealed Nodal-high tumor cells to be ALDH-low and vise versa, suggesting that Nodal may inhibit ALDH1 expression via stimulating TGF-ß signaling in uterine endometrioid adenocarcinoma. In fact, the addition of Nodal to endometrioid adenocarcinoma cell line reduced ALDH1-high population. Although ALDH1 mRNA level was not affected, the amount of ALDH1 protein appeared to be reduce by Nodal through ubiquitine-proteasome pathway. The regulation of TGF-ß signaling might be a novel therapeutic target of CICs in endometrioid adenocarcinoma.

Biosynthesis and degradation of canine placental prostaglandins: prepartum changes in expression and function of prostaglandin F2α-synthase (PGFS, AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD).

There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. Although the PGF2alpha-synthase (PGFS [AKR1C3]) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartments, the availability and biochemical activity of the PGFS (AKR1C3) protein remain unknown. In order to better understand the regulation of canine uterine PGF2alpha availability and eventual prepartum release in luteolytic amounts in dogs, canine-specific PGFS (AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD) antibodies were generated and used to characterize the expression, cellular localization, and biochemical properties of PGFS (AKR1C3) and HPGD in the utero/placental compartments and corpus luteum throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated postimplantation and declined prepartum. The utero/placental expression of PGFS (AKR1C3) was identified in the superficial uterine glands throughout gestation and in the trophoblast cells within the feto-maternal contact zone during placentation, suggesting a possible role for PGFS (AKR1C3) in the trophoblast invasion. Utero-placental HPGD was up-regulated until postimplantation, lower at midgestation, and greatly suppressed at prepartum. Expression was routinely identified in the endometrial surface and glandular epithelia, and positive signals were also observed in the trophoblast cells at the feto-maternal contact zone. The biochemical activity of recombinant PGFS (AKR1C3) and HPGD was confirmed after its expression in a heterologous system. The colocalization of HPGD with PGFS (AKR1C3) expression suggests a modulatory role for HPGD as a gatekeeper of the supply of prostaglandin in the pregnant canine uterus.

Alkaline phosphatases contribute to uterine receptivity, implantation, decidualization, and defense against bacterial endotoxin in hamsters.

Alkaline phosphatase (AP) activity has been demonstrated in the uterus of several species, but its importance in the uterus, in general and during pregnancy, is yet to be revealed. In this study, we focused on identifying AP isozyme types and their hormonal regulation, cell type, and event-specific expression and possible functions in the hamster uterus during the cycle and early pregnancy. Our RT-PCR and in situ hybridization studies demonstrated that among the known Akp2, Akp3, Akp5, and Akp6 murine AP isozyme genes, hamster uteri express only Akp2 and Akp6; both genes are co-expressed in luminal epithelial cells. Studies in cyclic and ovariectomized hamsters established that while progesterone (P4) is the major uterine Akp2 inducer, both P4 and estrogen are strong Akp6 regulators. Studies in preimplantation uteri showed induction of both genes and the activity of their encoded isozymes in luminal epithelial cells during uterine receptivity. However, at the beginning of implantation, Akp2 showed reduced expression in luminal epithelial cells surrounding the implanted embryo. By contrast, expression of Akp6 and its isozyme was maintained in luminal epithelial cells adjacent to, but not away from, the implanted embryo. Following implantation, stromal transformation to decidua was associated with induced expressions of only Akp2 and its isozyme. We next demonstrated that uterine APs dephosphorylate and detoxify endotoxin lipopolysaccharide at their sites of production and activity. Taken together, our findings suggest that uterine APs contribute to uterine receptivity, implantation, and decidualization in addition to their role in protection of the uterus and pregnancy against bacterial infection.

Successful implantation after reducing matrix metalloproteinase activity in the uterine cavity.

BACKGROUND: Recently, the concept of recurrent implantation failure (RIF) in assisted reproductive technology has been enlarged. Chronic uterine inflammation is a known cause of implantation failure and is associated with high matrix metalloproteinase (MMP) activity in uterine cavity flushing. MMP activity of women with RIF has been reported to be higher than that of fertile women. In the present retrospective study we evaluated the efficacy of treatment for high MMP activity in the uterine cavity of patients with RIF. METHODS: Of the 597 patients recruited to the study, 360 patients underwent MMP measurements and 237 patients did not (control group). All patients had failed to become pregnant, despite at least two transfers of good-quality embryos. Gelatinase MMP-2 and MMP-9 activity in uterine flushing fluid was detected by enzymology (MMP test). All samples were classified into two groups (positive or negative) based on the intensity of the bands on the enzyme zymogram, which represents the degree of MMP activity. Patients who tested positive on the initial test were treated for 2 weeks with a quinolone antibiotic and a corticosteroid, and subsequently underwent a second MMP test. Negative results on the second MMP tests after treatment and subsequent rates of pregnancy and miscarriage were used to evaluate the efficacy of treatment. Data were analyzed by the Mann-Whitney U-test and the chi-square test. RESULTS: Of the patients who underwent the MMP test, 15.6% had positive results (high MMP activity). After treatment, 89.3% of patients had negative results on the second MMP test. These patients had a significantly better pregnancy rate (42.0%) than the control group (26.6%), as well as a lower miscarriage rate (28.5% vs 36.5%, respectively). CONCLUSIONS: A 2-week course of antibiotics and corticosteroids effectively improves the uterine environment underlying RIF by reducing MMP activity.