Identification of highly reliable risk genes for Alzheimer's disease through joint-tissue integrative analysis.

Numerous genetic variants associated with Alzheimer's disease (AD) have been identified through genome-wide association studies (GWAS), but their interpretation is hindered by the strong linkage disequilibrium (LD) among the variants, making it difficult to identify the causal variants directly. To address this issue, the transcriptome-wide association study (TWAS) was employed to infer the association between gene expression and a trait at the genetic level using expression quantitative trait locus (eQTL) cohorts. In this study, we applied the TWAS theory and utilized the improved Joint-Tissue Imputation (JTI) approach and Mendelian Randomization (MR) framework (MR-JTI) to identify potential AD-associated genes. By integrating LD score, GTEx eQTL data, and GWAS summary statistic data from a large cohort using MR-JTI, a total of 415 AD-associated genes were identified. Then, 2873 differentially expressed genes from 11 AD-related datasets were used for the Fisher test of these AD-associated genes. We finally obtained 36 highly reliable AD-associated genes, including APOC1, CR1, ERBB2, and RIN3. Moreover, the GO and KEGG enrichment analysis revealed that these genes are primarily involved in antigen processing and presentation, amyloid-beta formation, tau protein binding, and response to oxidative stress. The identification of these potential AD-associated genes not only provides insights into the pathogenesis of AD but also offers biomarkers for early diagnosis of the disease.

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.

[HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway].

Objective: To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action. Methods: Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with HADHA overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT). Results: HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01,P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced. Conclusion: HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.

Exposure to Benzo(a)pyrene damages mitochondrial function via suppressing mitochondrial melatonin receptors in ovarian corpus luteum during early pregnancy.

Benzo(a)pyrene (BaP) is a well-known environmental endocrine pollutant, which has ovarian toxicity in mammals. Ovarian corpus luteum (CL), as the main source of progesterone synthesis in early pregnant female, requires a large number of mitochondria for energy supply. We previously demonstrated that BaP and its metabolite benzo(a)pyren-7, 8-dihydrodiol-9, 10-epoxide (BPDE) inhibited the ovarian melatonin receptors (MTRs) expression and decreased the levels of estrogen and progesterone during early pregnancy in mice. Emerging researches show that MTRs also exist on mitochondrial membrane and participate in the regulation of mitochondrial function. However, the relationship between BaP, MTRs on mitochondrial membrane and mitochondrial function remains unknown. Consequently, this study focuses on the effect and potential mechanism of BaP on ovarian luteal mitochondrial function during early pregnancy. We found that BaP and its metabolite BPDE decreased MTRs in early pregnant CL and luteinized KGN cells, especially in mitochondria. Furthermore, BaP or BPDE up-regulated the expression of SIRT3, Mfn2 and Drp-1, damaged mitochondrial morphology and decreased the MMP and the ATP levels, thereby causing mitochondrial dysfunction. Notably, activation of the MTRs on mitochondrial membrane by MTRs agonist ramelteon partially alleviated BPDE-induced up-regulation of SIRT3, Mfn2 and Drp-1, reduced mitochondrial fragmentation and enhanced the MMP and the ATP levels, thus restoring the expression of steroid rate-limiting enzymes. Together, these findings firstly proved that BaP and BPDE down-regulate MTRs on mitochondrial membrane, and further injure mitochondrial function in early pregnant rats' CL, which provides a new insight for understanding the exact mechanism of the BaP-induced ovarian toxicity.

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.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

The dual roles of three MMPs and TIMP in innate immunity and metamorphosis in the silkworm, Bombyx mori.

The matrix metalloproteinases (MMPs) and their endogenous inhibitory factors, tissue inhibitors of metalloproteinases (TIMPs), are implicated in many diseases. However, the mammalian MMPs (> 20) and TIMPs (> 3) are larger in number, and so little is known about their individual roles in organisms. Hence, we have systematically studied the roles of all three MMPs and one TIMP in silkworm innate immunity and metamorphosis. We observed that MMPs and TIMP are highly expressed during the pupation stage of the silkworms, and TIMP could interact with each MMPs. High-activity MMPs and low-activity TIMP may enhance the infection of B. mori nucleopolyhedrovirus in both in vitro and in vivo. MMPs' knockout and TIMP overexpression delayed silkworm development and even caused death. Interestingly, different MMPs' knockout led to different tubular tissue dysplasia. These findings provide insights into the conserved functions of MMPs and TIMP in human organogenesis and immunoregulation.

In utero Exposure to Excessive Estrogen Impairs Homologous Recombination and Oogenesis via Estrogen Receptor 2 in Mice.

The association between the accumulation of synthetic chemicals with estrogenic activity and risks to oogenesis has become a growing concern. This study indicates that in utero estrogen exposure can affect homologous recombination in early oogenesis and influence the reproductive potential and lifespan of female offspring. We conducted this study in developing mouse ovaries using two different models: oral doses administered to the mother, and fetal ovary cultures. Our analyses of meiotic fetal oocytes suggest that 17-ß-estradiol induces gross aberrations in prophase I events, including delayed meiotic progression, increased unrepaired DNA damage, and altered homologous recombination levels. These effects were mainly mediated by estrogen receptor 2 (ESR2) activation. Mid-gestation exposure to estrogen also led to delayed primordial folliculogenesis after birth, impaired follicle development after prepuberty, and ultimately reduced the total litter size of the offspring. This raises the concern that maternal exposures to substances activating ESR2 may compromise the fertility of the exposed female fetus.

High-fat diet-induced obesity primes fatty acid ß-oxidation impairment and consequent ovarian dysfunction during early pregnancy.

BACKGROUND: Obesity is associated with many adverse effects on female fertility. Obese women have a higher likelihood of developing ovulatory dysfunction due to dysregulation of the hypothalamic-pituitary-ovarian axis. However, the effect of obesity on ovarian function during early pregnancy needs to be further assessed. METHODS: C57BL6/J mice were given a high-fat diet (HFD) for 12 weeks to induce obesity. An in vitro high-fat model was established by treating the human ovarian granulosa cell line KGN with oleic acid and palmitic acid. Ovarian morphology of obese mice in early pregnancy was assessed by hematoxylin and eosin staining and ovarian function was assessed by enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. Oil Red O staining and transmission electron microscopy were used to detect fatty acid accumulation. Specific markers relating to the ovarian functional mechanism were assessed by real-time PCR, western blotting, lactate detection, adenosine triphosphate (ATP) detection, biochemical analyses, and enzyme-linked immunosorbent assay. RESULTS: The results of this study showed that during early pregnancy, the number of corpus lutea, serum estradiol and progesterone levels, and the expression of the steroid biosynthesis-related protein CYP19A1 (aromatase), CYP11A1 (cholesterol side chain cleavage enzyme), and StAR (steroidogenic acute regulatory protein), were significantly increased in HFD mice. Mice fed an HFD also showed a significant increase in ovarian lipid accumulation on day 7 of pregnancy. Genes involved in fatty acid synthesis (Acsl4 and Elovl5), and fatty acid uptake and transport (Slc27a4), together with the ß-oxidation rate-limiting enzyme Cpt1a, were significantly upregulated in HFD mice. Specifically, there was abnormal elevation of ATP and aberrant expression of tricarboxylic acid cycle (TCA)- and electron transport chain (ETC)-related genes in the ovaries of pregnant HFD mice. KGN cells treated with etomoxir targeting ß-oxidation of fatty acid showed decreased TCA cycle and ETC related gene expression. The elevation of ATP and estradiol and progesterone levels was reversed. CONCLUSIONS: During early pregnancy, HFD-induced obesity increases fatty acid ß-oxidation, which in turn increases TCA cycle and ETC related gene expression, leading to increased ATP production and ovarian dysfunction.

Exposure to butylated hydroxytoluene compromises endometrial decidualization during early pregnancy.

Butylated hydroxytoluene (BHT), one of the most widely used synthetic phenolic antioxidants, is a popular food additive. Previous studies have reported the possible health hazards of BHT. However, BHT effects on female reproduction, especially on endometrial decidualization, are still unknown. During early pregnancy, decidualization plays important roles for embryo implantation and pregnancy establishment. This study aimed to explore the effects of BHT on endometrial decidualization in pregnant mice. The pregnant mice received BHT via intraperitoneal injection at doses of 0, 200, and 400 mg/kg/day from day 1 (D1) of pregnancy until sacrifice. Under BHT exposure, maternal body weight was significantly decreased during early pregnancy. Compared with the control group, the number of implantation sites and uterine weight were significantly reduced in the BHT groups. The uterine lumen failed to close after BHT exposure, and the decidual morphology of endometrial stromal cells was inhibited by BHT. Furthermore, BHT significantly decreased the expression of endometrial decidual markers including COX2, HOXA10, and MMP9. Notably, the levels of serum estrogen (E2) and progesterone (P4) and expression levels of uterus estrogen receptor α (ERα) and progesterone receptor (PR) during early pregnancy were significantly upregulated following BHT exposure. In conclusion, these results demonstrated that gestational BHT exposure could inhibit decidualization of mouse endometrium during early pregnancy. The disorders of reproductive hormones and changes of hormone receptor signals could be responsible for the impaired decidualization. This study provided new evidence for the deleterious effects of BHT on female reproduction and revealed the potential reproductive toxicity of synthetic phenolic antioxidants.

Ephrin and Eph receptor signaling in female reproductive physiology and pathology†.

Ephrins are ligands of Eph receptors (Ephs); both of which are sorted into two classes, A and B. There are five types of ephrin-As (ephrin-A1-5) and three types of ephrin-Bs (ephrin-B1-3). Also, there are 10 types of EphAs (EphA1-10) and six types of EphBs (EphB1-6). Binding of ephrins to the Eph receptors activates signaling cascades that regulate several biological processes such as cellular proliferation, differentiation, migration, angiogenesis, and vascular remodeling. Clarification of their roles in the female reproductive system is crucial to understanding the physiology and pathology of this system. Such knowledge will also create awareness regarding the importance of these molecules in diagnostic, prognostic, and therapeutic medicine. Hence, we have discussed the involvement of these molecules in the physiological and pathological events that occur within the female reproductive system. The evidence so far suggests that the ephrins and the Eph receptors modulate folliculogenesis, ovulation, embryo transport, implantation, and placentation. Abnormal expression of some of these molecules is associated with polycystic ovarian syndrome, ovarian cancer, tubal pregnancy, endometrial cancer, uterine leiomyoma (fibroids), cervical cancer, and preeclampsia, suggesting the need to utilize these molecules in the clinical setting. To enhance a quick development of this gradually emerging field in female reproductive medicine, we have highlighted some "gaps in knowledge" that need prospective investigation.

Abnormal angiogenesis of placenta in progranulin­deficient mice.

Progranulin (PGRN) is a secreted growth factor involved in pleiotropic functions, particularly angiogenesis. A distinctly different placental expression of PGRN has been reported between normal pregnancies and pregnancies with complications, such as pre­eclampsia or fetal growth restriction. However, the role of PGRN in placental vascular development remains to be elucidated. In the present study, PGRN­knockout mice (PGRN­/­) were used to investigate the role of PGRN in the development of placental blood vessels and placental formation. Placental weights and pup body weights were significantly lower in the PGRN­/­ mice compared with the wild­type mice. Reduced labyrinthine layer areas and aberrant vascularization were also observed via hematoxylin and eosin staining of PGRN­/­ mice at embryonic day 14.5 (E14.5) and E17.5. In addition, the morphological data obtained via immunohistochemistry, immunofluorescence staining and western blotting demonstrated decreased expression levels of the blood vessel markers α­smooth muscle actin and CD31 in PGRN­/­ placentas. Furthermore, vasodilator endothelial nitric oxide synthase was reduced in the PGRN­/­ placenta. These results indicated that PGRN serves an essential role in the normal angiogenesis of the placental labyrinth in mice.

Stomatin-like protein 2 (SLP2) regulates the proliferation and invasion of trophoblast cells by modulating mitochondrial functions.

INTRODUCTION: Stomatin-like protein 2 (SLP2) is highly expressed in human first trimester trophoblast cells, but its functions in placental morpho-physiology remain unknown. This study aimed to determine the role of SLP2 in the proliferation and invasion of human first trimester trophoblast cells. METHODS: Immunofluorescence was used to determine the expression and localization of SLP2 in normal and miscarriage human first trimester placenta. Western blot was used to determine the expression of SLP2, PCNA, Cyclin D3, N-cadherin, Vimentin, PGC1α and PPARα in HTR-8/SVneo cells. SLP2 was knocked down in the HTR-8/SVneo cells by using si-Slp2. Wound healing and migration assays were used to determine the effect of SLP2 knockdown on the migration and invasion in the HTR-8/SVneo cells. Mitochondrial membrane potential, reactive oxygen species (ROS), ATP production and biogenesis were measured to assess the effects of SLP2 knockdown on mitochondrial functions. RESULT: SLP2 was strongly expressed in the cytotrophoblasts (CTB), syncytiotrophoblast (STB) and extravillous trophoblasts (EVT) of normal pregnancy placenta as compared to miscarriage placenta. SLP2 was highly expressed in the invasive EVT cell lines, HTR-8/SVneo and HPT-8 compared to the CTB cell line JAR. Knockdown of SLP2 significantly inhibited the migration and invasion of HTR-8/SVneo cells and placental villous explants, and repressed mitochondrial biogenesis and functions in HTR-8/SVneo cells. DISCUSSION: Silencing of SLP2 inhibited the proliferation, migration and invasion of HTR-8/SVneo cells via the impairment of mitochondrial functions. This indicates that the downregulation of SLP2 in miscarriage placenta could be part of the pathogenesis and pathophysiology of the disease.

Exposure to Benzo[a]pyrene impairs the corpus luteum vascular network in rats during early pregnancy.

Benzo [a]pyrene (BaP) is a well-known endocrine disruptor. Exposure to BaP is known to impair embryo implantation. The corpus luteum (CL), the primary source of progesterone during early pregnancy, plays a pivotal role in embryo implantation and pregnancy maintenance. The inappropriate luteal function may result in implantation failure and spontaneous abortions. However, the effect of BaP on CL remains unknown. This study investigated the deleterious effects of BaP on the structure and function of CL during early pregnancy. Pregnant rats were dosed with BaP at 0.2 mg.kg-1. d from day 1 (D1) to day 9 (D9) of gestation. We found that BaP reduced the number of CLs, disturbed the secretion of steroid and impacted the luteal vascular networks. BaP significantly decreased the angiogenesis factor (VEGFR, Ang-1 and Tie2) and increased the anti-angiogenic factor THBS1. Inhibited THBS1 function by LSKL partially rescued the angiogenesis defect caused by BaP. In vitro, BaP metabolite BPDE also interfered the expression levels of angiogenesis-related factors in HUVECs and impaired the angiogenesis, whereas supplemented with rAng-1 can alleviate the anti-angiogenic effect of BPDE. Furthermore, Notch signaling molecules, including Notch1, Dll4, Jag1 and Hey2, which are essential for the establishment and maturation of vascular networks, were affected by BaP exposure. Collectively, BaP broke the molecular regulatory balance between luteal angiogenesis and vascular maturation, impaired the construction of luteal vascular networks, and further affected luteal formation and endocrine function during early pregnancy. Our findings might provide new insight into the relationship between BaP and luteal insufficiency in early pregnancy. These data also give a new line of evidence for curtailing BaP emissions and protecting the women of childbearing age from occupational exposure.

Dibutyl phthalate exposure disrupts the progression of meiotic prophase I by interfering with homologous recombination in fetal mouse oocytes.

Dibutyl phthalate (DBP), one of the most widely used plasticizers, is a known environmental endocrine disruptor that impairs male and female fertility. In this study, oral administration of DBP was given to pregnant mice on 14.5 days post coitus (dpc) for 3 days; and additionally, DBP was added into the culture of 14.5 dpc fetal ovaries for 3 days. DBP exposure during gestation disturbed the progression of meiotic prophase I of mouse oocytes, specifically from the zygotene to pachytene stages. Meanwhile, the DBP-exposed pachytene oocytes showed increased homologous recombination sites and unrepaired DNA damage. Furthermore, DBP caused DNA damage by increasing oxidative stress, decreased the expression of multiple critical meiotic regulators, and consequently induced oocyte apoptosis. Moreover, the effect of DBP on meiosis I prophase involved estrogen receptors α and ß. Collectively, these results demonstrated a set of meiotic defects in DBP-exposed fetal oocytes. As aberrations in homologous recombination can result in aneuploid gametes and embryos, this study provides new support for the deleterious effects of phthalates.

Bombyx mori protein BmREEPa and BmPtchd could form a complex with BmNPV envelope protein GP64.

Our previous studies have indicated that Bombyx mori receptor expression enhancing protein a (BmREEPa) could participate in BV invasion in vivo and in vitro, however, the mechanism is still unclear. In this study, we screened BmREEPa interacting protein through co-immunoprecipitation and finally identified a membrane protein, Bombyx mori patched domain containing protein (BmPtchd, KR338939), which contains receptor activity. Further studies showed that BmPtchd, BmREEPa and Glycoprotein 64 could form a protein complex and the expression level of BmREEPa and BmPtchd could be affected by cellular cholesterol level. These findings may provide an important basis for explaining the invasion mechanism of Bombyx mori Nucleopolyhedrovirus budded virus.