Insulin-like growth factor II, a marker gene for determining the optimum dietary protein level in clownfish Amphiprion ocellaris.

In vivo study was conducted to determine the role of insulin-like growth factors (IGF-II) related to dietary protein utilization. For this early juvenile stage of marine false clownfish Amphiprion ocellaris, 300 numbers of 20-day-old larvae (initial body weight of 18.2±0.027 mg/fish) were used as an experimental animal. Animals were fed for 12 weeks with different dietary protein levels (35, 40, 45, 50, and 55%), using Spirulina maxima as a major protein source. Proximate compositions and amino acid profile of the formulated diet were analyzed by the standard methods. Eventually, significant (P<0.05) mean, body weight gain, absolute growth rate, specific growth rate, and feed conversion ratio were obtained in the fishes fed with 50% of dietary protein, whereas, poor growth performance was noticed in 35% of the dietary protein level fed juveniles. The growth-responsible gene IGF-II expression studies showed the significant upregulation in the growth of the juveniles at 2.05±0.11 for 40%, 3.13±0.20 for 45%, 4.97±0.13 for 50%, and 4.33± 0.24 for 55%, which were higher than the control group 35%. The study concluded that 50% of dietary protein level is optimal for better growth indices in clownfish; A. ocellaris juveniles and IGF-II can be used as a potential marker gene to assess the growth indices in A. ocellaris.

The Role of SOX9 in IGF-II-Mediated Pulmonary Fibrosis.

Pulmonary fibrosis (PF) associated with systemic sclerosis (SSc) results in significant morbidity and mortality. We previously reported that insulin-like growth factor-II (IGF-II) is overexpressed in lung tissues and fibroblasts from SSc patients, and IGF-II fosters fibrosis by upregulating collagen type I, fibronectin, and TGFß. We now show that IGF-II augments mRNA levels of profibrotic signaling molecules TGFß2 (p ≤ 0.01) and TGFß3 (p ≤ 0.05), collagen type III (p ≤ 0.01), and the collagen posttranslational modification enzymes P4HA2 (p ≤ 0.05), P3H2 (p ≤ 0.05), LOX (p = 0.065), LOXL2 (p ≤ 0.05), LOXL4 (p ≤ 0.05) in primary human lung fibroblasts. IGF-II increases protein levels of TGFß2 (p ≤ 0.01), as well as COL3A1, P4HA2, P4Hß, and LOXL4 (p ≤ 0.05). In contrast, IGF-II decreases mRNA levels of the collagen degradation enzymes cathepsin (CTS) K, CTSB, and CTSL and protein levels of CTSK (p ≤ 0.05). The SRY-box transcription factor 9 (SOX9) is overexpressed in SSc lung tissues at the mRNA (p ≤ 0.05) and protein (p ≤ 0.01) levels compared to healthy controls. IGF-II induces SOX9 in lung fibroblasts (p ≤ 0.05) via the IGF1R/IR hybrid receptor, and SOX9 regulates TGFß2 (p ≤ 0.05), TGFß3 (p ≤ 0.05), COL3A1 (p ≤ 0.01), and P4HA2 (p ≤ 0.001) downstream of IGF-II. Our results identify a novel IGF-II signaling axis and downstream targets that are regulated in a SOX9-dependent and -independent manner. Our findings provide novel insights on the role of IGF-II in promoting pulmonary fibrosis.

Embryonic growth retardation and altered expression of IGF-II is reciprocal induced by phytocompounds during early gestation in mice.

Methanolic crude extract of Scoparia dulcis (CESD) was orally administered to female mice during the early gestation (day 4-day 8) at a dose of 500 mg/kg/day. It induces embryo resorption and morphological changes of fetal maternal tissue. Histomorphology was studied by routine hematoxylin eosin stain. In situ immunofluorescence localization of IGF-II using Texas red showed an ordered expression of the growth factor in the maternal decidual cells, trophoblast cells and the embryo. Western blot analysis showed a gradual increase of IGF-II from D4 to D8 of control females. In contrast, the CESD-treated females showed resorption of embryo on D8 with disorganized in situ expression and lowered IGF-II in fetal maternal tissue. The phytocompounds present in the CESD could modulate either the ER or IGF-II receptors causing reduced IGF-II expression in the target tissues which lead to the failure of embryonic growth during periimplantation.

Tanshinone IIA inhibits Leu27IGF-II-induced insulin-like growth factor receptor II signaling and myocardial apoptosis via estrogen receptor-mediated Akt activation.

Different stress condition stimulates the expression level of insulin-like growth factor receptor II (IGF-IIR) in cardiomyoblasts that lead to apoptosis. Tanshinone IIA (TSN), a pharmacologically active component from Danshen, has been shown cardioprotective effects against cardiac apoptosis induced by several stress conditions. Therefore, this study was conducted to assess the cardioprotective effects of TSN IIA mediated through the estrogen receptor (ER) in order to inhibit the Leu27IGF-II-enhanced IGF-IIR-mediated cardiac apoptosis. The estrogenic activity of TSN IIA was examined after myocardial cells were pretreated with the ER antagonist, and inhibited the phospho-inositide-3 kinase (PI3K). Here, we found that TSN IIA significantly induced ER that phosphorylated Akt. Further, Akt activation considerably suppressed the Leu27IGF-II induced IGF-IIR expression level and the downstream effectors, including Gαq and calcineurin as well as mitochondrial dependent apoptosis proteins including Bad, cytochrome c, and active caspase-3 that result in cardiac apoptosis resistance. However, the western blot analysis, JC-1 staining, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay revealed that TSN IIA attenuated Leu27IGF-II-induced IGF-IIR mediated cardiac apoptosis was reversed by an ER antagonist such as ICI 182780, and PI3K inhibition. All these findings demonstrate that TSN IIA exerts estrogenic activity, which can activate PI3K-Akt pathway, and thereby inhibits Leu27IGFII induced IGF-IIR mediated cardiac apoptosis. Thus, TSN IIA can be considered as an effective therapeutic strategy against IGF-IIR signaling cascade to suppress cardiac apoptosis.

Disruption of the GHRH receptor and its impact on children and adults: The Itabaianinha syndrome.

Since 1994, we have been studying an extended kindred with 105 subjects (over 8 generations) residing in Itabaianinha County, in the Brazilian state of Sergipe, who have severe isolated GH deficiency (IGHD) due to a homozygous inactivating mutation (c.57 + 1G > A) in the GH releasing hormone (GHRH) receptor (GHRHR) gene. Most of these individuals have never received GH replacement therapy. They have low GH, and very low and often undetectable levels of serum IGF-I. Their principal physical findings are proportionate short stature, doll facies, high-pitched-voice, central obesity, wrinkled skin, and youthful hair with delayed pigmentation, and virtual absence of graying. The newborns from this cohort are of normal size, indicating that GH is not needed for intra-uterine growth. However, these IGHD individuals exhibit a myriad of phenotypic changes throughout the body, with a greater number of beneficial than harmful consequences. This GHRH signal disruption syndrome has been a valuable model to study the GH roles in body size and function. This reviews summarized the findings we have reported on this cohort.

Specific epigenetic microenvironment and the regulation of tumor-related gene expression by trichloroethylene in human hepatocytes.

Trichloroethylene (TCE), an important volatile organic solvent, causes a series of toxic damage to human. Conventional genetic mechanisms cannot fully explain its toxicity and carcinogenicity, indicative of the possible involvement of epigenetic mechanisms. Our study was intended to investigate the epigenetic toxicity and underlying mechanisms of TCE. Data showed that 0.3 mM TCE treatment for 24 h increased the growth of L-02 cells transiently. In contrast, subacute exposure to TCE inhibited cell growth and induced the genomic DNA hypomethylation and histone hyperacetylation. Further studies have revealed the TCE-induced DNA hypomethylation in the promoter regions of tumor-related genes, N-Ras, c-Jun, c-Myc, c-Fos and IGF-II, promoting their protein levels in a time-dependent manner. These results reveal there is a negative relationship existing between DNA hypomethylation and protein expression in tumor-related gene after TCE exposure under specific epigenetic microenvironment, serving as early biomarkers for TCE-associated diseases.

Immunohistochemical study of IGF-I and IGF-II expression in canine mammary tumours: Prognostic and diagnostic role.

Mammary tumours are among the most common tumours in dogs and are of interest due to their similarities to human breast tumours. Insulin-like growth factors (IGFs) are considered important in cell growth and development. The aim of this study was to investigate the immunohistochemical expression of IGF-I and IGF-II in benign and malignant canine mammary tumours. In this study, 10 benign and 10 malignant mammary tumours from the archives of the Department of Pathology were used, and five normal breast tissues were used as controls. It was observed that the expression of IGF-I and IGF-II was low to absent in benign tumours and increased in malignant tumours. The expression of IGF-II was higher than that of IGF-I. This study showed that IGF-I and IGF-II can be used as criteria for malignancy in canine mammary tumours. The results also indicate that IGF-I and IGF-II may be used as early diagnostic markers, and their inhibition may be used for the treatment of canine and human mammary tumours in the future.

Development of a Parallel Reaction Monitoring-MS Method To Quantify IGF Proteins in Dogs and a Case of Nonislet Cell Tumor Hypoglycemia.

Nonislet-cell tumor hypoglycemia (NICTH) is a rare paraneoplastic phenomenon well described in dogs and humans. Tumors associated with NICTH secrete incompletely processed forms of insulin-like growth factor-II (IGF-II), commonly named big IGF-II. These forms have increased bioavailability and interact with the insulin and IGF-I receptor causing hypoglycemia and growth-promoting effects. Immunoassays designed for human samples have been used to measure canine IGF-I and -II, but they possess some limitations. In addition, there are no validated methods for measurement of big IGF-II in dogs. In the present study, a targeted parallel reaction monitoring MS-based method previously developed for cats has been optimized and applied to simultaneously quantify the serum levels of IGF-I, IGF-II, and IGFBP-3, and for the first time, the levels of big IGF-II in dogs. This method allows the absolute quantification of IGF proteins using a mixture of QPrEST proteins previously designed for humans. The method possesses good linearity and repeatability and has been used to evaluate the IGF-system in a dog with NICTH syndrome. In this dog, the levels of big IGF-II decreased by 80% and the levels of IGF-I and IGFBP-3 increased approximately 20- and 4-times, respectively, after removal of the tumor.

Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. METHODS: Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. RESULTS: Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II-induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. CONCLUSION: Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms underlying HBV-related HCC and may lead to the development of effective therapies.

Roles of insulin-like growth factors in metamorphic development of turbot (Scophthalmus maximus).

Larval turbot (Scophthalmus maximus) undergo metamorphosis, a late post-embryonic developmental event that precedes juvenile transition. Insulin-like growth factors (IGFs) are important endocrine/autocrine/paracrine factors that provide essential signals to control of the embryonic and postnatal development of vertebrate species, including fish. Accumulating evidence suggests that IGFs are involved in regulating the metamorphic development of flatfish. This mini review focus on the functions of all known IGFs (IGF-I and IGF-II) during the metamorphic development of turbot. Information about IGFs and insulin-like growth factors binding proteins (IGFBPs) from other teleosts is also included in this review to provide an overview of IGFs functions in the metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGFs system in controlling of flatfish metamorphosis and contributing to the improvement of broodstock management strategies for larval turbot.

Silencing IGF-II impairs C-myc and N-ras expressions of SMMC-7721 cells via suppressing FAK/PI3K/Akt signaling pathway.

Emerging evidence confirms that insulin-like growth factor -II (IGF-II), oncogenes C-myc and N-ras are an essential regulator for development and growth in hepatocellular carcinoma (HCC). Although our previous study also indicated that IGF-II might upregulate levels of oncogenes C-myc and N-ras in hepatoma carcinoma cells, the molecular mechanism had not been fully elucidated. Herein, we successfully silenced IGF-II expression in SMCC-7721 cells by small RNA interference. Functional analysis showed that knockdown of IGF-II significantly suppressed growth and proliferation of SMMC-7721 cells and decreased C-myc and N-ras mRNA and protein levels. And this function was mediated by the FAK/PI3K/Akt signaling pathway. Taken together, IGF-II siRNA inactivates the FAK/PI3K/Akt signaling pathway, and further reduces cell proliferation, N-ras and C-myc levels in SMMC-7721 cells. Especially, understanding the relationship between IGF-II and oncogenes N-ras and C-myc in cancer cells will provide novel clues for clinic HCC treatment in the future.

IGF-II-mediated downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α in myoblast cells involves PI3K/Akt/FoxO1 signaling pathway.

Insulin-like growth factor II (IGF-II) can stimulate myogenesis and is critically involved in skeletal muscle differentiation. The presence of negative regulators of this process, however, is not well explored. Here, we showed that in myoblast cells, IGF-II negatively regulated peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA expression, while constitutive expression of PGC-1α induced myoblast differentiation. These results suggest that the negative regulation of PGC-1α by IGF-II may act as a negative feedback mechanism in IGF-II-induced myogenic differentiation. Reporter assays demonstrated that IGF-II suppresses the basal PGC-1α promoter activity. Blocking the IGF-II signaling pathway increased the endogenous PGC-1α levels. In addition, pharmacological inhibition of PI3 kinase activity prevented the downregulation of PGC-1α but the activation of mTOR was not required for this process. Importantly, further analysis showed that forkhead transcription factor FoxO1 contributes to mediating the effects of IGF-II on PGC-1 promoter activity. These findings indicate that IGF-II reduces PGC-1α expression in skeletal muscle cells through a mechanism involving PI3K-Akt-FoxO1 but not p38 MAPK or Erk1/2 MAPK pathways.

Association of tRNA methyltransferase NSUN2/IGF-II molecular signature with ovarian cancer survival.

AIM: To investigate the association between NSUN2/IGF-II signature and ovarian cancer survival. METHODS: Using a publicly accessible dataset of RNA sequencing and clinical follow-up data, we performed Classification and Regression Tree and survival analyses. RESULTS: Patients with NSUN2 high IGF-II low had significantly superior overall and disease progression-free survival, followed by NSUN2 low IGF-II low, NSUN2 high IGF-II high and NSUN2 low IGF-II high (p < 0.0001 for overall, p = 0.0024 for progression-free survival, respectively). The associations of NSUN2/IGF-II signature with the risks of death and relapse remained significant in multivariate Cox regression models. Random-effects meta-analyses show the upregulated NSUN2 and IGF-II expression in ovarian cancer versus normal tissues. CONCLUSION: The NSUN2/IGF-II signature associates with heterogeneous outcome and may have clinical implications in managing ovarian cancer.

The behavioral deficits and cognitive impairment are correlated with decreased IGF-II and ERK in depressed mice induced by chronic unpredictable stress.

AIM: To investigate the behavioral deficits, cognitive impairment and possible mechanisms induced by chronic unpredictable stress (CUS) in mice of different ages. METHODS: The behaviors and cognition were tested using the open field test, tail suspension test and the Morris water maze. The changes in the insulin-like growth factor II (IGF-II) and the extracellular signal-regulated kinase (ERK) and phosphorylation (p-ERK) in the hippocampus (HP) and prefrontal cortex (PFC) were evaluated by immunohistochemistry. RESULTS: The 15-month-old mice revealed a significant decline in spontaneous behavior and the learning-memory function and showed a decrease in IGF-II and p-ERK expression levels in HP and PFC. Four weeks of CUS exposure induced significant anxiety- and depression-like behavior and learning-memory function impairment in 3- to 15-month-old mice, and reduced IGF-II and p-ERK expression levels in HP and PFC, compared with control group mice, respectively. CONCLUSIONS: The behavioral deficits and cognitive impairment induced by CUS and aging in mice could be associated with the down-regulated expression of IGF-II and p-ERK in HP and PFC. This role seems to be dependent on the intracellular ERK pathway.

LIN-28B/let-7a/IGF-II axis molecular subtypes are associated with epithelial ovarian cancer prognosis.

OBJECTIVES: Aberrant expressions of LIN-28B, let-7a and IGF-II occur in epithelial ovarian cancer, and the LIN-28B/let-7a/IGF-II axis is associated with human disease. The purpose of this study was to investigate the associations between LIN-28B/let-7a/IGF-II axis molecular subtypes and epithelial ovarian cancer prognosis. METHODS: Using quantitative reverse transcription PCR, we analyzed LIN-28B, let-7a and IGF-II mRNA in 211 primary epithelial ovarian cancer tissues, and also performed Classification and Regression Tree (CART) and survival analyses. RESULTS: Four terminal subtypes were identified in the CART analysis in combination with survival analysis. Kaplan-Meier survival curves showed that subtypes LIN-28B(low)let-7a(low) and LIN-28B(low)let-7a(high)IGF-II(low) had significantly better survival than subtypes LIN-28B(high) or LIN-28B(low)let-7a(high)IGF-II(high) (p<0.0001 for overall, p=0.017 for progression-free survival, respectively). Multivariate Cox regression models showed that compared to subtype LIN-28B(high), subtypes LIN-28B(low)let-7a(low) and LIN-28B(low)let-7a(high)IGF-II(low) had significantly reduced mortality and reduced relapse risks. Moreover, subtype LIN-28B(low)let-7a(low) had better response to chemotherapy than subtype LIN-28B(high). CONCLUSIONS: These results suggest that molecular subtypes of the LIN-28B/let-7a/IGF-II axis associate with heterogeneous progression and may have clinical implications in predicting epithelial ovarian cancer prognosis.

Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae.

Insulin-like growth factor-II regulates bone sialoprotein gene transcription.

Insulin-like growth factor-I and -II (IGF-I and IGF-II) have been found in bone extracts of several different species, and IGF-II is the most abundant growth factor stored in bone. Bone sialoprotein (BSP) is a noncollagenous extracellular matrix glycoprotein associated with mineralized connective tissues. In this study, we have investigated the regulation of BSP transcription by IGF-II in rat osteoblast-like ROS17/2.8 cells. IGF-II (50 ng/ml) increased BSP mRNA and protein levels after 6-h stimulation, and enhanced luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Effects of IGF-II were inhibited by tyrosine kinase, extracellular signal-regulated kinase1/2 and phosphatidylinositol 3-kinase inhibitors, and abrogated by 2-bp mutations in cAMP response element (CRE), FGF2 response element (FRE) and homeodomain protein-binding site (HOX). The results of gel shift assays showed that nuclear proteins binding to CRE, FRE and HOX sites were increased by IGF-II (50 ng/ml) at 3 and 6 h. CREB1, phospho-CREB1, c-Fos and c-Jun antibodies disrupted the formation of the CRE-protein complexes. Dlx5 and Runx2 antibodies disrupted the FRE- and HOX-protein complex formations. These studies therefore demonstrated that IGF-II increased BSP transcription by targeting CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB1, c-Fos, c-Jun, Dlx5 and Runx2 transcription factors appear to be key regulators of IGF-II effects on BSP transcription.

Insulin-Like Growth Factor II (IGF-II) Inhibits IL-1ß-Induced Cartilage Matrix Loss and Promotes Cartilage Integrity in Experimental Osteoarthritis.

Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1ß-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1ß, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro. In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA.

Prenatal cocaine exposure impairs cognitive function of progeny via insulin growth factor II epigenetic regulation.

Studies have showed that prenatal cocaine exposure (PCOC) can impair cognitive function and social behavior of the offspring; however, the mechanism underlying such effect is poorly understood. Insulin-like growth factor II (Igf-II), an imprinted gene, has a critical role in memory consolidation and enhancement. We hypothesized that epigenetic regulation of hippocampal Igf-II may attribute to the cognitive deficits of PCOC offspring. We used Morris water maze and open-field task to test the cognitive function in PCOC offspring. The epigenetic alteration involved in hippocampal Igf-II expression deficit in PCOC offspring was studied by determining Igf-II methylation status, DNA methyltransferases (DNMT) expressions and L-methionine level. Moreover, IGF-II rescue experiments were performed and the downstream signalings were investigated in PCOC offspring. In behavioral tests, we observed impaired spatial learning and memory and increased anxiety in PCOC offspring; moreover, hippocampal IGF-II mRNA and protein expressions were significantly decreased. Hippocampal methylation of cytosine-phospho-guanine (CpG) dinucleotides in differentially methylated region (DMR) 2 of Igf-II was elevated in PCOC offspring, which may be driven by the upregulation of L-methionine and DNA methyltransferase (DNMT) 1. Importantly, intra-hippocampal injection of recombinant IGF-II reactivated the repressed calcium calmodulin kinase II α (CaMKIIα) and reversed cognitive deficits in PCOC offspring. Collectively, our findings suggest that cocaine exposure during pregnancy impairs cognitive function of offspring through epigenetic modification of Igf-II gene. Enhancing IGF-II signaling may represent a novel therapeutical strategy for cocaine-induced cognitive impairment.

Paternal breed effects on expression of IGF-II, BAK1 and BCL2-L1 in bovine preimplantation embryos.

The effects of the paternal breed on early embryo and later pre- and postnatal development are well documented. Several recent studies have suggested that such paternal effects may be mediated by the paternally induced epigenetic modifications during early embryogenesis. The objective of this study was to investigate the effects of the paternal breed on the early embryonic development and relative expression of the maternally imprinted gene, IGF-II, and the apoptosis-related genes BAK1 and BCL2-L1 in in vitro produced (IVP) bovine embryos derived from two unrelated paternal breeds (Holstein and Brown Swiss). The degree of correlation of IGF-II expression pattern with embryo developmental competence and apoptosis-related genes was also investigated. The relative abundance of IGF-II, BCL2-L1 and BAK1 transcripts in day 8 embryos was measured by quantitative reverse-transcription polymerase chain reaction using the comparative Cp method. Our data revealed that the paternal breed did not influence cleavage rate, blastocyst rate and relative abundance of IGF-II, BAK1 and BCL2-L1 in day 8 blastocysts (P > 0.05). Nevertheless, IGF-II expression levels were highly correlated with embryonic developmental competence (r = 0.66, P < 0.1), relative expression of BCL2-L1 (r = 0.72, P < 0.05) and ratio of BCL2-L1/BAK1 (r = 0.78, P < 0.05). In conclusion, our data show that IGF-II, BCL2-L1 and BAK1 expression is not related to the chosen combination of paternal breed, but that IGF-II expression is correlated with embryonic viability and apoptosis-related gene expression.