Tumor Necrosis Factor Alpha-induced Protein 6 Research Articles

Role of tumor necrosis factor alpha-induced protein 6 (TNFAIP6) in tumors: A pan-cancer analysis

Abstract Background There is growing evidence that the gene named tumor necrosis factor α–induced protein 6 (TNFAIP6) has an important role in various tumors. However, a systematic pan-cancer analysis of TNFAIP6 is lacking. Here we aimed to analyze the expression of TNFAIP6 across multiple cancers and verify its expression during the progression of colon cancer. Methods We performed a comprehensive bioinformatics analysis to examine the expression of TNFAIP6 across 27 tumor types. GEPIA2 was used to evaluate the effect of TNFAIP6 on clinical cancer prognosis. cBioportal was used to assess TNFAIP6 mutations. The correlation between TNFAIP6 and cancer immune infiltrates was explored using TIMER2.0. The CancerSEA database was used to perform functional analysis of TNFAIP6. Metascape was used to identify TNFAIP6-related gene enrichment pathways. Immunohistochemistry was performed to detect TNFAIP6 protein expression in the colon cancer. In addition, the Comparative Toxicogenomics Database was searched for known and possible antitumor drugs that may be associated with TNFAIP6. Results We found that, in most of the cancers included in this analysis, TNFAIP6 was highly expressed, and there is a distinct relationship between TNFAIP6 expression and cancer prognosis. TNFAIP6 expression is associated with cancer-associated fibroblasts, neutrophils, and endothelial cells. TNFAIP6 and similar genes may also be involved in the PID_VEGF_VEGFR_ pathway. Immunohistochemistry revealed an increasing trend of TNFAIP6 protein expression in normal, adenoma, and colon cancer tissues. Several known and possible antitumor drugs that may be associated with TNFAIP6 were identified in the Comparative Toxicogenomics Database. These results suggest that a number of drugs may target TNFAIP6 during cancer treatment, including cisplatin, irinotecan, resveratrol, U 0126, NSC689534, genistein, NSC668394, oxaliplatin, plerixafor, topotecan, vincristine, flutamide, doxorubicin, MRK 003, folic acid, demecolcine, tunicamycin, zoledronic acid, and schizandrin B. Conclusions TNFAIP6 may function as an oncogene in certain cancers. Furthermore, this study provides evidence that TNFAIP6 is an important factor in colon cancer progression.

TNFAIP6 defines the MSC subpopulation with enhanced immune suppression activities

BackgroundMesenchymal stromal/stem cells (MSCs) have been intensively investigated in both pre-clinical and clinical studies. However, the therapeutic efficacy varies resulting from the heterogenicity of MSCs. Therefore, purifying the specific MSC subpopulation with specialized function is necessary for their therapeutic applications.MethodsThe large-scale RNA sequencing analysis was performed to identify potential cell markers for the mouse MSCs. Then, the immune suppression activities of the purified MSC subpopulation were assessed in vitro and in vivo.ResultsThe TNFAIP6 (tumor necrosis factor alpha-induced protein 6) has been identified as a potential cell marker for mouse MSCs, irrespective of tissue origin and laboratory origin. The TNFAIP6+ mouse MSCs showed enhanced immune suppression activities and improved therapeutic effects on the mouse model of acute inflammation, resulting from faster response to immune stimulation.ConclusionsTherefore, we have demonstrated that the TNFAIP6+ MSC subpopulation has enhanced immune suppression capabilities.

P-651 Oocyte secreted factors supress the expression of prostaglandin synthase-2 (PTGS2), a key maturation promoter in cumulus cells: the oocyte is not in a hurry

Abstract Study question Does the oocyte control the expression of genes regulating the ovulatory cascade and the formation of the extracellular matrix in cumulus cells? Summary answer Oocyte secreted factors (OSFs) suppress the expression of prostaglandin synthase-2 (PTGS2) in cumulus cells, a key mediator of the maturation cascade. What is known already Oocyte quality depends on cytoplasmic and nuclear maturation synchrony. OSFs modulate maturation dynamics by inhibiting the expression of AREG (amphiregulin) and EREG (epiregulin), key mediators of the ovulatory cascade. AREG and EREG stimulate PTGS2 expression in cumulus cells, which catalyses the synthesis of prostaglandins, that amplify and translate AREG/EREG signals leading to meiotic completion and expression of genes required for cumulus expansion, among which HAS2 (hyaluronan synthase-2), essential for the synthesis of hyaluronan, the backbone of cumulus extracellular matrix, as well as PTX3 (pentraxin 3) and TNFAIP6 (tumor necrosis factor alpha-induced protein 6), genes encoding for matrix structural proteins. Study design, size, duration Relative mRNA levels of PTGS2, HAS2, PTX3 and TNFAIP6 were compared in cumulus cells from 3 treatment-groups: 1-COC: Intact cumulus-oocyte complexes subjected to in vitro maturation (IVM); 2-OOX: oocytectomized COC subjected to IVM; 3-OOX+DO: OOX subjected to IVM with the addition of denuded oocytes (1 DO/µL). Four experimental replicates including all three treatment-groups were performed. The inclusion of OOX+DO aimed to assess whether the effects of the oocyte on gene expression are mediated by OSFs. Participants/materials, setting, methods COC were aspirated from 3-8mm follicles of bovine ovaries. Oocytectomy was achieved by ooplasm aspiration with a micromanipulator. Pools of 20 COCs/OOX/OOX+DO were cultured for 22 hours in TCM199 with 100ng/mL AREG and 0.4% BSA. Subsequently, cumulus cells were separated, total RNA extracted, and mRNA abundance assessed by real-time RT-PCR with oligo-dT primers and two internal controls for relative quantification. Treatment effects were tested by ANOVA and groups were compared with the Tukey test. Main results and the role of chance Oocyctectomy drastically increased PTGS2 mRNA abundance, which was reversed by the addition of denuded oocytes (DO) to culture. Relative PTGS2 mRNA levels (mean ± EPM) were 1.02 ± 0.12, 10.40 ± 1.65 and 0.88 ± 0.08 for COC, OOX and OOX+DO, respectively (P = 0.02). Consistently, although in a lower magnitude, oocytectomy increased mRNA levels of PTX3(1.00 ± 0.04, 2.50 ± 0.18 and 0.75 ± 0.15 for COC, OOX and OOX+DO, respectively. P < 0.001) and TNFAIP6 (1.00 ± 0.07, 2.04 ± 0.17 and 0.67 ± 0.04 for COC, OOX and OOX+DO, respectively. P < 0.001), an effect again reversed by the presence of DO in culture. In contrast, oocytectomy decreased the expression of HAS2, which was neutralised in the presence of DO (1.01 ± 0.09, 0.69 ± 0.02 and 1.11 ± 0.08 for COC, OOX and OOX+DO, respectively. P = 0.005). Our data indicate that the oocyte modulates its own maturation pace by inhibiting the expression of PTGS2, which is accompanied by similar negative impacts on genes that strongly respond to prostaglandin signalling such as PTX3 and TNFAIP6. In parallel, our data suggest that OSFs exert a potent stimulatory effect on HAS2 transcription, capable to overcome their concomitant inhibitory influence via supressed AREG/EREG and PTGS2 expression. Limitations, reasons for caution Our study utilised animal and culture models that may not exactly represent human COC physiological maturation. In addition, our data are restricted to mRNA levels. Nevertheless, somatic cells predominantly transcribe poly A-mRNA that is promptly exported and translated. Accordingly, our RT-PCR strategy utilised poly-A reverse transcription with oligo-dT primers. Wider implications of the findings Our data indicate that OSFs inhibit the expression of PTGS2, a key gene for activation and amplification of the maturation cascade in cumulus cells. We speculate that this may represent an effort of the oocyte to prevent precocious meiotic resumption, thus favouring nuclear and cytoplasmic maturation synchrony and developmental competence. Trial registration number Not applicable

Compromised Cumulus-Oocyte Complex Matrix Organization and Expansion in Women with PCOS

The cumulus-oocyte complex (COC) matrix plays a critical role in the ovulation and fertilization process and a major predictor of oocyte quality. Proteomics studies of follicular fluid showed differential expression of COC matrix proteins in women with polycystic ovary syndrome (PCOS), indicating altered COC matrix in these women. In the present study, we aimed to understand COC matrix gene induction in humans and its probable dysfunction in women with PCOS. Animal studies have shown that amphiregulin (AREG) and growth differentiation factor-9 (GDF-9) are important in the induction of COC matrix genes which are involved in cumulus expansion. The effects of AREG and GDF-9 on expression of tumor necrosis factor alpha induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) on human cumulus granulosa cells (CGCs) and murine COC expansion were evaluated. Further time-dependent effects of growth factor supplementation on these gene expressions in CGCs from PCOS and control women were compared. Follicular fluid from PCOS showed reduced COC matrix expansion capacity, using murine COCs. Expression of COC matrix genes TNFAIP6 and HAS2 were significantly reduced in CGCs of PCOS. Treatment of CGCs with AREG and GDF-9 together induced expression of both these genes in controls and could only restore HAS2 but not TNFAIP6 expression in PCOS. Our results suggest that the reduced potential of follicular fluid to support COC expansion, altered expression of structural constituents, and intrinsic defects in granulosa cells of women with PCOS may contribute to the aberrant COC organization and expansion in PCOS, thus affecting fertilization.

Sex-specific reduction in inflammation of osteoarthritic human chondrocytes and nutraceutical-dependent extracellular matrix formation

IntroductionThe aim of this study was to investigate the ability of osteoarthritic human chondrocytes to produce articular cartilage (AC) tissues with a reduced inflammatory environment in response to 4 anti-inflammatory nutraceuticals: alpha-tocopherol (Alpha), gallic acid (G), ascorbic acid (AA), and catechin hydrate (C). MethodsChondrocytes isolated from patients who underwent total knee arthroplasty surgeries were divided into groups (9 male; mean age, 66.2 ± 3.5 years and 11 female; mean age, 64.2 ± 3.1 years). Cells were cultured based on sex and supplemented with either a negative control (NC) medium or NC plus one of the nutraceuticals at a concentration of 50 μM. At day 21, cultures were characterized histologically, biochemically, and for gene expression of vital markers. ResultsAt day 21, 62.3% and 66.2% reduction in nitric oxide (NO) content was evident for female and male cells, respectively. G-treatment of female cells resulted in the lowest expression of nitric oxide synthase-2 (NOS2), matrix metalloproteinase-13 (MMP13), and collagen type-10 (COL10). Alpha-treatment of male cells resulted in the lowest expression of NOS2, bone morphogenic protein-2, MMP13, COL10 and tumor necrosis factor alpha induced protein-6 (TNFAIP6) relative to NC. AA and Alpha treatment resulted in the highest glycosaminoglycan (GAG) content for female and male cultures, respectively. ConclusionA sex-dependent response of osteoarthritic chondrocytes to nutraceutical treatment was evident. Our results suggest the use of G for female cells and Alpha for male cells in OA applications seems to be favorable in reducing inflammation and enhancing chondrocytes’ ability to form AC tissues.

Functional Characterization of Fusion Proteins of the Tumor Necrosis Factor Alpha‐Induced Protein 6 (TNFAIP6) Domains

TNFAIP6 is an extracellular protein that is essential for cumulus cell extracellular matrix formation and female fertility in mice. A mammalian expression system was used to generate recombinant proteins of the full length TNFAIP6 and its individual domains, Link and CUB. These proteins contained a short amino terminal sequence essential for catalytic function and a carboxy‐terminal Fc region of mouse IgG2a for easy detection, and were termed TNFAIP6Fc, LinkFc and CUBFc, respectively. Hyaluronan‐binding of the fusion proteins was measured with an enzyme‐linked immunosorbent assay. The catalytic activity of the proteins was assessed by their ability to remove heavy chain 1 (HC1) from inter‐alpha‐trypsin inhibitor (IαI) using western‐blot. The ability of the fusion proteins to restore cumulus cell extracellular matrix formation was tested in a cell culture assay of Tnfaip6−/− cumulus cell‐oocyte complexes (COCs). Both TNFAIP6Fc and LinkFc were able to bind hyaluronan, with LinkFc showing much stronger binding at equimolar concentrations. CUBFc showed no significant binding to hyaluronan even at high concentrations. TNFAIP6Fc catalysed the removal of HC1 from IαI in a concentration dependent manner, indicating that the Fc tag did not interfere with catalytic function. LinkFc, CUBFc, and their combination were unable to remove the HC1 from IαI at high concentrations even though they contained the essential short amino‐terminal region. TNFAIP6Fc successfully restored cumulus cell extracellular matrix formation in Tnfaip6−/− COCs, while LinkFc, CUBFc, and their combination were unable to do so. LinkFc, CUBFc, and their combination did not disrupt normal cumulus cell extracellular matrix formation of Tnfaip6+/− COCs. Our results suggest that the catalytic function of TNFAIP6 requires both of its domains in addition to the previously identified serine residue in the amino‐terminal region, because neither domain alone is able to remove HC1 from IαI or support cumulus cell extracellular matrix formation in Tnfaip6−/− COCs. Furthermore, the absence of catalytic activity of both domains when combined together could imply a pivotal conformational interaction normally achieved by their covalent attachment. The finding that the LinkFc has stronger hyaluronan binding than TNFAIP6Fc could support the presence of such a conformational interaction that weakens the hyaluronan‐binding of the full‐length protein compared to the Link domain alone.Support or Funding InformationSupported by internal funding of Touro University Nevada

Systemic recovery and therapeutic effects of transplanted allogenic and xenogenic mesenchymal stromal cells in a rat blunt chest trauma model

BackgroundEffective therapy of Acute Lung Injury (ALI) is still a major scientific and clinical problem. To define novel therapeutic strategies for sequelae of blunt chest trauma (TxT) like ALI/Acute Respiratory Distress Syndrome, we have investigated the immunomodulatory and regenerative effects of a single dose of ex vivo expanded human or rat mesenchymal stromal cells (hMSCs/rMSCs) with or without priming, immediately after the induction of TxT in Wistar rats. MethodsWe analyzed the histological score of lung injury, the cell count of the broncho alveolar lavage fluid (BAL), the change in local and systemic cytokine level and the recovery of the administered cells 24 h and 5 days post trauma. ResultsThe treatment with hMSCs reduced the injury score 24 h after trauma by at least 50% compared with TxT rats without MSCs. In general, TxT rats treated with hMSCs exhibited a lower level of pro-inflammatory cytokines (interleukin [IL]-1B, IL-6) and chemokines (C-X-C motif chemokine ligand 1 [CXCL1], C-C motif chemokine ligand 2 [CCL2]), but a higher tumor necrosis factor alpha induced protein 6 (TNFAIP6) level in the BAL compared with TxT rats after 24 h. Five days after trauma, cytokine levels and the distribution of inflammatory cells were similar to sham rats. In contrast, the treatment with rMSCs did not reveal such therapeutic effects on the injury score and cytokine levels, except for TNFAIP6 level. ConclusionTxT represents a suitable model to study effects of MSCs as an acute treatment strategy after trauma. However, the source of MSCs has to be carefully considered in the design of future studies.

The CXCL12-CXCR4 signaling promotes oocyte maturation by regulating cumulus expansion in sheep

Gonadotropins and growth factors synergistically regulate folliculogenesis and oocyte development. C-X-C motif chemokine 12 (CXCL12) and its receptor CXCR4 are expressed in ovaries of sheep, cattle and other species, however, roles of this multifunctional signal axis in oocyte maturation are not defined. Using sheep as a model, we examined the expression patterns and functions of the CXCL12-CXCR4 axis during oocyte maturation. CXCL12 and CXCR4 mRNA and protein were present in oocytes and granulosa cells. Relative abundance of CXCR4 transcript was controlled by epidermal growth factor (EGF). Transient inhibition of CXCR4 suppressed oocyte nuclear maturation while supplementing recombination CXCL12 significantly increased percent of oocyte undergone metaphase I phase. Inhibition of CXCR4 function decreased cumulus expansion growth rate. Furthermore, granulosa cell migration was impaired and expression of hyaluronan synthase 2 (HAS2) and hyaluronan binding protein tumor necrosis factor-alpha-induced protein 6 (TNFAIP6) were downregulated by CXCR4 inhibition. These findings revealed a novel role of the CXCL12-CXCR4 signaling in oocyte development in sheep.

Reversible meiotic arrest of bovine oocytes by EGFR inhibition and follicular hemisections

The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 μM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 μM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.

Intrinsic multipotential mesenchymal stromal cell activity in gelatinous Heberden's nodes in osteoarthritis at clinical presentation.

IntroductionGelatinous Heberden’s nodes (HNs), also termed synovial cysts, are a common form of generalized osteoarthritis (OA). We sought to determine whether HN cases at clinical presentation contained multipotential stromal cells (MSCs) and to explore whether such cells were more closely related to bone marrow (BM) or synovial fluid (SF) MSCs by transcriptional analysis.MethodsAt clinical presentation, gelatinous material was extracted/extruded from the distal phalangeal joint of OA patients with HNs. From this, plastic adherent cells were culture-expanded for phenotypic and functional characterization and comparison with BM- and SF-MSCs. Mesenchymal related gene expression was studied by using a custom-designed TaqMan Low Density Array to determine transcriptional similarities between different MSC groups and skin fibroblasts.ResultsIn all cases, HN material produced MSC-like colonies. Adherent cultures displayed an MSC phenotype (CD29+, CD44+, CD73+, CD81+, and CD90+ and CD14- CD19-, CD31-, CD34-, CD45-, and HLADR-) and exhibited osteogenic, chondrogenic lineage differentiation but weak adipogenesis. Gene cluster analysis showed that HN-MSCs were more closely related to SF- than normal or OA BM-MSCs with significantly higher expression of synovium-related gene markers such as bone morphogenic protein 4 (BMP4), bone morphogenetic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled-related protein 4 (SFRP4), and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) (P <0.05).ConclusionsGelatinous HNs derived from hand OA at clinical presentation contain a population of MSCs that share transcriptional similarities with SF-derived MSCs. Their aberrant entrapment within the synovial cysts may impact on their normal role in joint homeostasis.

Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium

To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC). Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling. A reproductive biology laboratory. Not applicable. Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib. Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA. In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM). Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium.

2 TRANSCRIPTOME ANALYSIS OF GRANULOSA CELLS FROM GROWING DOMINANT FOLLICLE REVEALS AGE-ASSOCIATED CHANGES AT THE TIME OF FOLLICLE SELECTION IN AGED BEEF CATTLE

Growing dominant follicles at the time of selection from aged (n = 3; 15 ± 1.5 years) and young (n = 3; 6 ± 1.1 years) Hereford cows were compared using bovine-specific microarrays containing 40 000 targets. The objective of the study was to determine age-associated changes in transcriptome of granulosa cells at the time of dominant follicle selection. Cows were given prostaglandin F2α to cause ovulation (Day 0) and granulosa cells from dominant follicles were collected on Day 3 either by ultrasound-guided follicle aspiration or after ovariectomy. The mRNA was extracted, analyzed for quality, converted into antisense RNA, amplified, labelled with red and green florescent dyes, and hybridized with microarrays. Feature intensities were measured using Array-Pro software (Media Cybernetics Inc., Rockville, MD), and differentially expressed genes were obtained using FlexArray 1.6. A total of 169 transcripts were differentially expressed with a fold change of ≥2 (P ≤ 0.05) in aged cows v. young cows. Ingenuity System Pathway (IPA; Ingenuity Systems Inc., Redwood City, CA) analysis of these transcripts revealed that granulosa cells of aged cows exhibit (1) reduced capability to regulate gonadotropins [↓follistatin (FST), ↓inhibin beta A (INHBA), ↓inhibin beta B (INHBB)] and reduced responsiveness to gonadotropin-induced changes in cytoskeleton [↑tropomyosin 2 (TPM2), ↑actin gamma 2 (ACTG2), ↓tubulin beta] and extracellular matrix [↓tumor necrosis factor alpha-induced protein 6 (TNFAIP6), ↓versican (VCAN)], (2) inefficiency in processing lipids [↓low-density lipoprotein receptor (LDLR), ↓stearoyl-coenzyme A desaturase (SCD), ↑cluster of differentiation 36 (CD36), ↓sterol-C4-methyl oxidase-like (SC4MOL)] and synthesizing steroids [↓cytochrome P450, family 19, subfamily A, polypeptide 1 (CYP19A1), ↓cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1)], (3) decreased proliferation [↓proliferating cell nuclear antigen (PCNA)] and control of cell cycle check points [↓checkpoint kinase 1 (CHEK1), ↓centromere-associated protein E (CENPE)] and have poor intercellular communication [↓gap junction protein alpha 1 (GJA1)], and (4) higher expression of oxidative stress responsive genes [↑vanin-1 (VNN1), ↑vanin-2 (VNN2), and ↑glutathione peroxidase 3 (GPX3)]. A total of 6 transcripts: CYP19A1 (aromatase; P ≤ 0.1), VNN1 (P ≤ 0.05), INHBA (P ≥ 0.1), PCNA (P ≤ 0.001), TPM2 (P ≤ 0.1), and GJA1 (P ≤ 0.05) were selected to validate the microarray results via quantitative real-time PCR. In conclusion, granulosa cells of growing dominant follicles exhibit age-related changes in the transcriptome at the time of selection relative to young cows; changes that may explain follicle-associated loss of oocyte competence in aged cows. Research was supported by the Natural Sciences and Engineering Research Council of Canada (Ottawa, ON, Canada).

Dynamics of the transcriptome in the primate ovulatory follicle

Experiments were designed to evaluate changes in the transcriptome (mRNA levels) in the ovulatory, luteinizing follicle of rhesus monkeys, using a controlled ovulation model that permits analysis of the naturally selected, dominant follicle at specific intervals (0, 12, 24 and 36 h) after exposure to an ovulatory (exogenous hCG) stimulus during the menstrual cycle. Total RNA was prepared from individual follicles (n= 4-8/timepoint), with an aliquot used for microarray analysis (Affymetrix Rhesus Macaque Genome Array) and the remainder applied to quantitative real-time PCR (q-PCR) assays. The microarray data from individual samples distinctly clustered according to timepoints, and ovulated follicles displayed markedly different expression patterns from unruptured follicles at 36 h. Between timepoint comparisons revealed profound changes in mRNA expression profiles. The dynamic pattern of mRNA expression for steroidogenic enzymes (CYP17A, CYP19A, HSD3B2, HSD11B1 and HSD11B2), steroidogenic acute regulatory protein (StAR) and gonadotrophin receptors [LH/choriogonadotrophin receptor (LHCGR), FSH receptor (FSHR)] as determined by microarray analysis correlated precisely with those from blinded q-PCR assays. Patterns of mRNA expression for epidermal-growth-factor-like factors (amphiregulin, epiregulin) and processes [hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6 (TNFAIP6)] implicated in cumulus-oocyte maturation/expansion were also comparable between assays. Thus, several mRNAs displayed the expected expression pattern for purported theca (e.g. CYP17A), granulosa (CYP19A, FSHR), cumulus (HAS2, TNFAIP6) cell and surface epithelium (HSD11B)-related genes in the rodent/primate pre-ovulatory follicle. This database will be of great value in analyzing molecular and cellular pathways associated with periovulatory events in the primate follicle (e.g. follicle rupture, luteinization, inflammatory response and angiogenesis), and for identifying novel gene products controlling mammalian fertility.

Expression of Tumor Necrosis Factor Alpha-Induced Protein 6 Messenger RNA in Porcine Preovulatory Ovarian Follicles

It has been shown previously that tumor necrosis factor alpha-induced protein 6 (TNFAIP6) is essential for formation of the cumulus extracellular matrix and female fertility. Therefore, we studied the expression of TNFAIP6 mRNA in porcine preovulatory follicles. In addition, we asked whether the expression of TNFAIP6 mRNA changes in mural granulosa cells (MGCs) during the periovulatory period or after culture of oocyte-cumulus complexes (OCCs) or MGCs in vitro. Mural granulosa cells obtained from follicles on day 12 (D 12) and day 15 (D 15) of the estrous cycle, eCG-stimulated follicles, follicles at 4-32 h after hCG stimulation and MGCs and OCCs obtained from immature gilts and cultured for 0-44 h in vitro with gonadotropins were used for extraction of total RNA and assessment of the relative abundance (RA) of TNFAIP6 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The levels of TNFAIP6 mRNA were low in the follicles on D 12 and D 15 of the estrous cycle and at 66 h after eCG stimulation but were significantly increased at 4 h after hCG. The high level of TNFAIP6 expression was maintained until 16 h after hCG stimulation and gradually decreased at 24 and 32 h after hCG. During in vitro culture, FSH/LH-induced TNFAIP6 mRNA was expressed in both OCCs and MGCs in a similar temporal pattern as seen in vivo. We conclude that TNFAIP6 expression in the pig, like other species, increases in preovulatory follicles following the LH (hCG) surge. The OCC and MGC display similar patterns of TNFAIP6 expression under both in vivo and in vitro conditions.

Synthesis of Tumor Necrosis Factor Alpha-Induced Protein 6 in Porcine Preovulatory Follicles: A Study with A38 Antibody1

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.

Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators1

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients

Purpose. To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. Methods. Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. Results. Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. Conclusions. The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.

Maternal nutrition and the sex ratio at birth: River, J. P. & M. A. Crawford: Nature, [formula omitted]: 297, 1974. Nuffield Institute of Comparative Medicine, Zoological Society of London, Regent's Park, London NW1 4RY, England

The objective of this study was to investigate the effects of inhibiting the epidermal growth factor receptor (EGFR) pathway on meiosis blockage and resumption, mRNA expression of genes involved in oocyte maturation and cumulus expansion, and embryo development. Bovine cumulus-oocyte complexes (COCs) were cultured for 15 h in the presence of the EGFR inhibitor (AG1478) and follicular hemisections (FHS). Most of the oocytes (89.3%) remained at the germinal vesicle (GV) stage when cultured in the presence of FHS and 5 μM AG1478. The inhibitory effect was reversible as most oocytes (83.8%) completed meiosis after additional 20 h maturation. Embryo development to the blastocyst stage was similar (P > 0.05) between FHS and 5 μM AG1478 treated (39.3%) and control (41.1%) groups. In cumulus cells, mRNA abundance of early growth response protein 1 (EGR1), tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and hyaluronan synthase 2 (HAS2) genes, and phosphorylated extracellular regulated kinase (p-ERK1/2) protein were lower in COCs treated with AG1478 plus FHS compared with FHS alone (P < 0.05). In granulosa cells of FHS, AG1478 treatment reduced transcript levels of PGR and ADAMTS1 (P < 0.05). The inhibitory effect of AG1478 on meiotic progression was not reverted by treatment with angiotensin II (ANG II) or prostaglandins (PGF2α or PGE2). This study demonstrates that inhibition of EGFR in the presence of FHS is a reliable approach to promote reversible arrest of bovine oocytes at the GV stage.