Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility.

DNA double-strand breaks (DSBs) are functionally linked to genomic instability in spermatocytes and to male infertility. The heavy metal cadmium (Cd) is known to induce DNA damage in spermatocytes by unknown mechanisms. Here, we showed that Cd ions impaired the canonical non-homologous end-joining (NHEJ) repair pathway, but not the homologous recombination (HR) repair pathway, through stimulation of Ser2056 and Thr2609 phosphorylation of DNA-PKcs at DSB sites. Hyper-phosphorylation of DNA-PKcs led to its premature dissociation from DNA ends and the Ku complex, preventing recruitment of processing enzymes and further ligation of DNA ends. Specifically, this cascade was initiated by the loss of PP5 phosphatase activity, which results from the dissociation of PP5 from its activating ions (Mn), that is antagonized by Cd ions through a competitive mechanism. In accordance, in a mouse model Cd-induced genomic instability and consequential male reproductive dysfunction were effectively reversed by a high dosage of Mn ions. Together, our findings corroborate a protein phosphorylation-mediated genomic instability pathway in spermatocytes that is triggered by exchange of heavy metal ions.

B(a)P induces ovarian granulosa cell apoptosis via TRAF2-NFκB-Caspase1 axis during early pregnancy.

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H2O2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency.

Identification and Validation of Tumor Microenvironment-Associated Signature in Clear-Cell Renal Cell Carcinoma through Integration of DNA Methylation and Gene Expression.

The tumor microenvironment (TME) is crucial in tumor development, metastasis, and response to immunotherapy. DNA methylation can regulate the TME without altering the DNA sequence. However, research on the methylation-driven TME in clear-cell renal cell carcinoma (ccRCC) is still lacking. In this study, integrated DNA methylation and RNA-seq data were used to explore methylation-driven genes (MDGs). Immune scores were calculated using the ESTIMATE, which was employed to identify TME-related genes. A new signature connected with methylation-regulated TME using univariate, multivariate Cox regression and LASSO regression analyses was developed. This signature consists of four TME-MDGs, including AJAP1, HOXB9, MYH14, and SLC6A19, which exhibit high methylation and low expression in tumors. Validation was performed using qRT-PCR which confirmed their downregulation in ccRCC clinical samples. Additionally, the signature demonstrated stable predictive performance in different subtypes of ccRCC. Risk scores are positively correlated with TMN stages, immune cell infiltration, tumor mutation burden, and adverse outcomes of immunotherapy. Interestingly, the expression of four TME-MDGs are highly correlated with the sensitivity of first-line drugs in ccRCC treatment, especially pazopanib. Molecular docking indicates a high affinity binding between the proteins and pazopanib. In summary, our study elucidates the comprehensive role of methylation-driven TME in ccRCC, aiding in identifying patients sensitive to immunotherapy and targeted therapy, and providing new therapeutic targets for ccRCC treatment.

Nuclear erythroid 2-related factor 2 protects against reactive oxygen species -induced preterm premature rupture of membranes through regulation of mitochondria†.

Preterm premature rupture of membranes (pPROM) is a major cause of preterm birth and neonatal mortality. Reactive oxygen species (ROS) have been identified as a critical factor in the development of pPROM. Mitochondria are known to be the primary source of ROS and play a vital role in maintaining cellular function. The Nuclear erythroid 2-related factor 2 (NRF2) has been demonstrated to play a crucial role in regulating mitochondrial function. However, research exploring the impact of NRF2-regulated mitochondria on pPROM is limited. Therefore, we collected fetal membrane tissues from pPROM and spontaneous preterm labor (sPTL) puerpera, measured the expression level of NRF2, and evaluated the degree of mitochondrial damage in both groups. In addition, we isolated human amniotic epithelial cells (hAECs) from the fetal membranes and used small interfering RNA (siRNA) to suppress NRF2 expression, enabling us to evaluate the impact of NRF2 on mitochondrial damage and ROS production. Our findings indicated that the expression level of NRF2 in pPROM fetal membranes was significantly lower than in sPTL fetal membranes, accompanied by increased mitochondrial damage. Furthermore, after the inhibition of NRF2 in hAECs, the degree of mitochondrial damage was significantly exacerbated, along with a marked increase in both cellular and mitochondrial ROS levels. The regulation of the mitochondrial metabolic process via NRF2 in fetal membranes has the potential to influence ROS production.

Glycyrrhizin ameliorates impaired glucose metabolism and ovarian dysfunction in a polycystic ovary syndrome mouse model.

The aim of this study was to determine the impact of glycyrrhizin, an inhibitor of high mobility group box 1, on glucose metabolic disorders and ovarian dysfunction in mice with polycystic ovary syndrome. We generated a polycystic ovary syndrome mouse model by using dehydroepiandrosterone plus high-fat diet. Glycyrrhizin (100 mg/kg) was intraperitoneally injected into the polycystic ovary syndrome mice and the effects on body weight, glucose tolerance, insulin sensitivity, estrous cycle, hormone profiles, ovarian pathology, glucolipid metabolism, and some molecular mechanisms were investigated. Increased number of cystic follicles, hormonal disorders, impaired glucose tolerance, and decreased insulin sensitivity in the polycystic ovary syndrome mice were reverted by glycyrrhizin. The increased high mobility group box 1 levels in the serum and ovarian tissues of the polycystic ovary syndrome mice were also reduced by glycyrrhizin. Furthermore, increased expressions of toll-like receptor 9, myeloid differentiation factor 88, and nuclear factor kappa B as well as reduced expressions of insulin receptor, phosphorylated protein kinase B, and glucose transporter type 4 were restored by glycyrrhizin in the polycystic ovary syndrome mice. Glycyrrhizin could suppress the polycystic ovary syndrome-induced upregulation of high mobility group box 1, several inflammatory marker genes, and the toll-like receptor 9/myeloid differentiation factor 88/nuclear factor kappa B pathways, while inhibiting the insulin receptor/phosphorylated protein kinase B/glucose transporter type 4 pathways. Hence, glycyrrhizin is a promising therapeutic agent against polycystic ovary syndrome.

Emodin improves glucose metabolism and ovarian function in PCOS mice via the HMGB1/TLR4/NF-κB molecular pathway.

In brief: Obese PCOS mice display metabolic and endocrine disorders that manifest as abnormal metabolism of glucose and dysfunctions in the reproductive system. This study demonstrates that emodin alleviates most of these conditions possibly via the HMGB1/TLR4/NF-kB pathway. Abstract: PCOS is a reproductive disorder with an unclear etiology. It affects 5-10% of women worldwide and is largely associated with impaired glucose metabolism and obesity. HMGB1 is a nuclear protein associated with impaired glucose metabolism and PCOS. We sought to investigate the potential therapeutic effects of emodin on glucose metabolism and ovarian functions in PCOS mice via the HMGB1 molecular pathway. A high-fat diet (HFD) and dehydroepiandrosterone (DHEA)- induced PCOS mouse model comprising four experimental groups was established: control, PCOS, PCOS plus emodin, and PCOS plus vehicle groups. Emodin administration attenuated obesity, elevated fasting glucose levels, impaired glucose tolerance, and insulin resistance, and improved the polycystic ovarian morphology of PCOS mice. Additionally, it lowered elevated serum HMGB1, LH, and testosterone levels in PCOS mice. Elevated ovarian protein and mRNA levels of HMGB1 and TLR4 in PCOS mice were also lowered following emodin treatment. Furthermore, emodin lowered high NF-ĸB/65 protein levels in the ovaries of PCOS mice. Immunohistochemical staining of the ovaries revealed strong HMGB1, TLR4, and AR expressions in PCOS mice, which were lowered by emodin treatment. Moreover, emodin significantly increased GLUT4, IRS2, and INSR levels that were lowered by PCOS. Overall, our study showed that emodin alleviated the impaired glucose metabolism and improved ovarian function in PCOS mice, possibly via the HMGB1/TLR4/NF-ĸB signaling pathway. Thus, emodin could be considered a potential therapeutic agent in the management of PCOS.

Uterine deficiency of Dnmt3b impairs decidualization and causes consequent embryo implantation defects.

Uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects. Recent advances in molecular technologies have allowed the unprecedented mapping of epigenetic modifications during embryo implantation. DNA methyltransferase 3a (DNMT3A) and DNMT3B are responsible for establishing DNA methylation patterns produced through their de novo-type DNA methylation activity in implantation stage embryos and during germ cell differentiation. It was reported that conditional knockout of Dnmt3a in the uterus does not markedly affect endometrial function during embryo implantation, but the tissue-specific functions of Dnmt3b in the endometrium during embryo implantation remain poorly understood to investigate the role of Dnmt3b during peri-implantation period. Here, we generated Dnmt3b conditional knockout (Dnmt3bd/d) female mice using progesterone receptor-Cre mice and examined the role of Dnmt3b during embryo implantation. Dnmt3bd/d female mice exhibited compromised fertility, which was associated with defective decidualization, but not endometrial receptivity. Furthermore, results showed loss of Dnmt3b did not lead to altered genomic methylation patterns of the decidual endometrium during early pregnancy. Transcriptome sequencing analysis of uteri from day 6 pregnant mice identified phosphoglycerate kinase 1 (Pgk1) as one of the most variable genes in Dnmt3bd/d decidual endometrium. Potential roles of PGK1 in the decidualization process during early pregnancy were confirmed. Lastly, the compromised decidualization upon the downregulation of Dnmt3b could be reversed by overexpression of Pgk1. Collectively, our findings indicate that uterine deficiency of Dnmt3b impairs decidualization and consequent embryo implantation defects.

GIMICA: host genetic and immune factors shaping human microbiota.

Besides the environmental factors having tremendous impacts on the composition of microbial community, the host factors have recently gained extensive attentions on their roles in shaping human microbiota. There are two major types of host factors: host genetic factors (HGFs) and host immune factors (HIFs). These factors of each type are essential for defining the chemical and physical landscapes inhabited by microbiota, and the collective consideration of both types have great implication to serve comprehensive health management. However, no database was available to provide the comprehensive factors of both types. Herein, a database entitled 'Host Genetic and Immune Factors Shaping Human Microbiota (GIMICA)' was constructed. Based on the 4257 microbes confirmed to inhabit nine sites of human body, 2851 HGFs (1368 single nucleotide polymorphisms (SNPs), 186 copy number variations (CNVs), and 1297 non-coding ribonucleic acids (RNAs)) modulating the expression of 370 microbes were collected, and 549 HIFs (126 lymphocytes and phagocytes, 387 immune proteins, and 36 immune pathways) regulating the abundance of 455 microbes were also provided. All in all, GIMICA enabled the collective consideration not only between different types of host factor but also between the host and environmental ones, which is freely accessible without login requirement at: https://idrblab.org/gimica/.

Exposure to Benzo(a)pyrene promotes proliferation and inhibits differentiation of stromal cells in mice during decidualization.

The environmental pollutant Benzo(a)pyrene (BaP) has an adverse effect on the reproductive performance of mammals. We previously showed that BaP treatment during early pregnancy damages endometrial morphology and impairs embryo implantation. Endometrial decidualization at the implantation site (IS) after embryo implantation is crucial for pregnancy maintenance and placental development. The balance between proliferation and differentiation in endometrial stromal cells (ESCs) is a crucial event of decidualization, which is regulated by the cell cycle. Here, we report that abnormal decidualization caused by BaP is associated with cell cycle disturbance of stromal cells. The mice in the treatment group were gavaged with 0.2 mg/kg/day BaP from day 1-8 of pregnancy, while those in control were gavaged with corn oil in parallel. BaP damaged the decidualization of ESCs and reduced the number of polyploid cells. Meanwhile, BaP up-regulated the expression of Ki67 and PCNA, affecting the differentiation of stromal cells. The cell cycle progression analysis during decidualization in vivo and in vitro showed that BaP induced polyploid cells deficiency with enhanced expressions of CyclinA(E)/CDK2, CyclinD/CDK4 and CyclinB/CDK1, which promote the transformation of cells from G1 to S phase and simultaneously activate the G2/M phase. The above results indicated that BaP exposure accelerates cell cycle progression, promotes ESC proliferation, inhibits differentiation, and impedes proper decidualization and polyploidy development. Thus, the imbalance of ESC proliferation and differentiation would be an important mechanism for BaP-induced defective decidualization.

A decrease in cluster of differentiation 2 expression on natural killer cells is associated with polycystic ovary syndrome but not influenced by metformin in a mouse model†.

PROBLEM: Natural killer (NK) cells from the peripheral blood and spleen represent the source from which various tissues replenish their immune cell populations. Hyperandrogenism and high interleukin-2 (IL-2) levels are factors present in polycystic ovary syndrome (PCOS). These factors and metformin, one of the commonest medications used in treating PCOS, may have an impact on NK cells. However, this is presently unknown. Here, we aimed to assess the distribution of peripheral blood and splenic NK cells and their CD2 and CD94 expression patterns in a PCOS mouse model and test whether metformin could reverse these effects. METHOD OF STUDY: Four mouse groups were designed as follows (n = 15/group): control, PCOS, PCOS plus vehicle, PCOS plus metformin. Dehydroepiandrosterone and a high-fat diet were administered to induce the PCOS mouse model. Flow cytometry was used to analyze the expressions of CD2 and CD94 on peripheral blood and splenic NK cells. RESULTS: PCOS mice had a low surface-density of CD2 on peripheral blood NK cells and a decreased percentage of CD2+ splenic NK cells. Metformin administration did not significantly influence these changes; however, it reduced the splenic NK cell counts. CONCLUSIONS: Our findings proved the association of PCOS with an altered expression of CD2 on peripheral blood and splenic NK cells and that of metformin with a lowered splenic NK cell reserve in PCOS conditions. These findings could further unlock key mechanisms in PCOS pathophysiology and in the mechanism of action of metformin, towards improving PCOS management.

The roles of ADAMDEC1 in trophoblast differentiation during normal pregnancy and preeclampsia.

Human cytotrophoblast (CTB) differentiation into syncytiotrophoblast (STB) is essential for placental formation and function. Understanding the molecular mechanisms involved in trophoblast differentiation is necessary as it would help in the development of novel therapeutic agents to treat placentation-mediated pregnancy complications. In this study, we found a common upregulated gene, ADAM-like Decysin-1 (ADAMDEC1), from five published microarray and RNA-sequencing datasets. Interference to ADAMDEC1 impaired forskolin-induced BeWo cells differentiation, while ADAMDEC1 overexpression promoted BeWo cells and 3D JEG-3 spheroids differentiation. Interestingly, ADAMDEC1 may inhibit Thrombospondin 1 rather than E-cadherin to trigger the activation of the cAMP signal pathway during CTB differentiation into STB. More importantly, a decreasing in ADAMDEC1 might be involved in the development of preeclampsia. Therefore, ADAMDEC1 is expected to become a new target for prediction of and intervention in placenta-derived pregnancy diseases.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

Rictor/mTORC2 is involved in endometrial receptivity by regulating epithelial remodeling.

Successful embryo implantation requires well-functioning endometrial luminal epithelial cells to establish uterine receptivity. Inadequate uterine receptivity is responsible for approximately two thirds of implantation failures in humans. However, the regulatory mechanism governing this functional process remains largely unexplored. A previous study revealed that the expression of Rictor, the main member of mTORC2, in mouse epithelial cells is increased on the fourth day of gestation (D4). Here, we provide the first report of the involvement of Rictor in the regulation of endometrial receptivity. Rictor was conditionally ablated in the mouse endometrium using a progesterone receptor cre (PRcre ) mouse model. Loss of Rictor altered polarity remodeling and the Na+ channel protein of endometrial cells by mediating Rac-1/PAK1(pPAK1)/ERM(pERM) and Sgk1/pSgk1 signaling, respectively, ultimately resulting in impaired fertility. In the endometrium of women with infertility, the expression of Rictor was changed, along with the morphological transformation and Na+ channel protein of epithelial cells. Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity in both mice and humans. The present study may help improve the molecular regulatory network of endometrial receptivity and provide new diagnostic and treatment strategies for infertility.

Ovarian inflammatory mRNA profiles of a dehydroepiandrosterone plus high-fat diet-induced polycystic ovary syndrome mouse model.

RESEARCH QUESTION: What is the expression pattern of inflammatory mRNA profiles of a dehydroepiandrosterone (DHEA) plus high-fat diet (HFD)-induced polycystic ovary syndrome (PCOS) mouse model? DESIGN: RNA sequencing was performed to investigate the mRNA expression profiles in the ovarian tissues of a DHEA plus HFD-induced PCOS mouse model. Six samples were divided into two groups (control and PCOS), with three biological replicates in each group. This was followed by hierarchical clustering, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. The relative expression levels of nine inflammatory genes were validated via quantitative reverse-transcription polymerase chain reaction. RESULTS: A total of 436 genes were differentially expressed between the control and PCOS mice. Out of these, 137 genes were up-regulated while 299 genes were down-regulated. Gene ontology analysis indicated that differentially expressed mRNA were associated with T cell-mediated cytotoxicity and homocysteine metabolic processes. Pathway analysis further showed that these abnormally expressed mRNA were associated with signalling pathways, such as NF-kB signalling, tyrosine metabolism and phenylalanine metabolism. All these pathways are involved in chronic inflammation and PCOS. CONCLUSION: The differentially expressed genes are potentially involved in the inflammation that is evident in PCOS, and so could serve as therapeutic options against the disease. Nevertheless, prospective studies are needed to test this hypothesis.

Acidity scales of deep eutectic solvents based on IR and NMR.

Acidic deep eutectic solvents (ADESs) have been utilized in various applications. Clearly, it is crucial to obtain acidity information that could reveal the relationship with performance. However, appropriate methods for measuring acidity are limited. Herein, we developed two promising approaches (without additional solvents) to identify and characterize both Lewis and Brønsted acidities by applying acetonitrile as an infrared probe and trimethylphosphine oxide (TMPO) as a nuclear magnetic resonance (NMR) probe. The acetonitrile IR approach is suitable for measuring the acidity of Lewis ADESs by monitoring the peak of ν(CN) around 2300 cm-1, and the 31P-TMPO NMR approach could identify and scale both Lewis and Brønsted acidities precisely. Moreover, a perfect linear relationship between the IR shift of ν(CN) and the effective charge density of metal cations was established, which provides a better understanding of Lewis acidity. In short, this study not only offers two efficient acidity measurement methods but also provides a molecular basis for optimizing the performance of ADESs in applications.

The role of fascin in carcinogenesis and embryo implantation.

The cytoskeleton, with its actin bundling proteins, plays crucial roles in a host of cellular function, such as cancer metastasis, antigen presentation and trophoblast migration and invasion, as a result of cytoskeletal remodeling. A key player in cytoskeletal remodeling is fascin. Upregulation of fascin induces the transition of epithelial phenotypes to mesenchymal phenotypes through complex interaction with transcription factors. Fascin expression also regulates mitochondrial F-actin to promote oxidative phosphorylation (OXPHOS) in some cancer cells. Trophoblast cells, on the other hand, exhibit similar physiological functions, involving the upregulation of genes crucial for its migration and invasion. Owing to the similar tumor-like characteristics among cancer and trophoblats, we review recent studies on fascin in relation to cancer and trophoblast cell biology; and based on existing evidence, link fascin to the establishment of the maternal-fetal interface.

Downregulation of fascin in the first trimester placental villi is associated with early recurrent miscarriage.

Inadequate trophoblast proliferation, shallow invasion and exaggerated rate of trophoblast apoptosis are implicated in early recurrent miscarriage (ERM). However, the mechanistic bases of this association have not been fully established. We aimed at investigating the involvement of fascin, an actin-bundling protein, in trophoblast activities and ERM. We found that fascin was downregulated in the cytotrophoblasts (CTBs) and distal cytotrophoblasts (DCTs) of ERM placentae. Knockdown of fascin altered cellular and nucleolar morphology, and inhibited the proliferation but increased apoptosis of trophoblastic HTR8/SVneo cells. Furthermore, fascin knockdown decreased the expression of transcription factors such as Snail1/2, Twist and Zeb1/2, mesenchymal molecules such as Vimentin and N-cadherin, and the protein expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylates signal transducer and activator of transcript 3 (STAT3). Exposure of HTR-8/SVneo cells to hypoxia reoxygenation (H/R) decreased fascin expression to affect the cells' invasion. Our results indicate for the first time that the downregulation of fascin is involved in the pathogenesis of early recurrent miscarriage; and hence a potential therapeutic target against the disease.

Impairment of endometrial decidual reaction in early pregnant mice fed with high fat diet.

OBJECTIVE: To investigate the effect of obesity induced by high fat diet on decidual reaction of endometrium in mice, and the effect of high fat treatment on decidual reaction of endometrial stromal cells. METHODS: Twelve 4-week-old healthy C57BL/6J female mice were randomly divided into high fat diet group and control group with 6 mice in each group. They were fed with high fat diet (22 kJ/g) or normal diet (16 kJ/g) for 12 weeks, respectively. The body weight of mice was measured every week. After feeding for 12 weeks, the body length and width of mice were measured, and the levels of fasting serum triglyceride and total cholesterol were determined. Then the mice were mated with healthy C57BL/6J male mice, and the uterine tissues were collected on the seventh day of pregnancy. The decidual cells and collagen fibers in mouse endometrium was observed by HE staining and Masson staining respectively. The expression of decidual reaction related proteins in mouse endometrium were detected by immunohistochemistry and Western blotting. Mouse endometrial stromal cells (mESCs) were isolated and treated with the oleic acid and palmitic acid in vitro, and the decidual reaction was induced with estradiol and progesterone. The accumulation of lipid droplets in mESCs was observed by oil red O and Bodipy staining. The cytoskeleton of mESCs was observed by phalloidin staining. The levels of decidual reaction related genes and proteins were detected by real-time fluorescence quantitative PCR and Western blotting. RESULTS: After feeding for 12 weeks, the body weight of mice in the high fat group was significantly higher than that in the control group ( P<0.01), and there was no significant difference in body length between two groups ( P>0.05), but the body width of mice in the high fat group was significantly larger than that in the control group ( P<0.01), and the levels of serum triglyceride and total cholesterol were significantly higher than those in the control group (Both P<0.05). The number of embryo implantation in the high fat group was significantly less than that in the control group ( P<0.01). The differentiation of mESCs to decidual cells in high fat group was slow and abnormal. The expression levels of decidual reaction markers bone morphogenetic protein (BMP)2 and homeobox A10 (HOXA10) were lower than those in the control group, and there was significant difference in the expression level of HOXA10 ( P<0.01). The results of oil red O and Bodipy staining in mESCs showed that after high fat treatment, the accumulation of lipid droplets increased significantly, phalloidin staining showed abnormal cytoskeleton morphology. The expression levels of decidual reaction related genes dtprp, HOXA10 and proteins BMP2, HOXA10 and cyclooxygenase (COX)2 were significantly lower than those in the control group ( P<0.05). CONCLUSION: Obesity induced by high fat diet and high fat treatment can impair the decidual reaction of endometrium and endometrial stromal cells in mice.

Proline-rich 11 (PRR11) drives F-actin assembly by recruiting the actin-related protein 2/3 complex in human non-small cell lung carcinoma.

The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100-184 or 100-200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.

AMPK/mTOR downregulated autophagy enhances aberrant endometrial decidualization in folate-deficient pregnant mice.

Existing evidence suggests that adverse pregnancy outcomes are closely related to dietary factors. Folate plays an important role in neural tube formation and fetal growth, folate deficiency is a major risk factor of birth defects. Our early studies showed that folate deficiency could impair enddecidualization, however, the mechanism is still unclear. Dysfunctional autophagy is associated with many diseases. Here, we aimed to evaluate the adverse effect of folate deficiency on endometrial decidualization, with a particular focus on endometrial cell autophagy. Mice were fed with no folate diet in vivo and the mouse endometrial stromal cell was cultured in a folate-free medium in vitro. The decrease of the number of endometrial autophagosomes and the protein expressions of autophagy in the folate-deficient group indicated that autophagosome formation, autophagosome-lysosome fusion, and lysosomal degradation were inhibited. Autophagic flux examination using mCherry-GFP-LC3 transfection showed that the fusion of autophagosomes with lysosomes was inhibited by folate deficiency. Autophagy inducer rapamycin could reverse the impairment of folate deficiency on endometrial decidualization. Moreover, folate deficiency could reduce autophagy by disrupting AMPK/mTOR signaling, resulting in aberrant endometrial decidualization and adverse pregnancy outcomes. Further co-immunoprecipitation examination showed that decidual marker protein Hoxa10 could interact with autophagic marker protein Cathepsin L, and the interaction was notably reduced by folate deficiency. In conclusion, AMPK/mTOR downregulated autophagy was essential for aberrant endometrial decidualization in early pregnant mice, which could result in adverse pregnancy outcomes. This provided some new clues for understanding the causal mechanisms of birth defects induced by folate deficiency.