Leukaemia inhibitory factor in implantation and uterine biology

Leukaemia inhibitory factor (LIF) is one of the most important cytokines in the reproductive tract. Without expression of LIF in the uterus, implantation of a blastocyst cannot begin. Yet, 13 years after publication of the phenotype of the LIF knockout mouse we are only just beginning to understand how LIF functions in the uterus. This review addresses our knowledge of the role of LIF in regulating implantation through its influence on the luminal epithelium and stromal decidualization, but also its influence on reproductive tract cells such as leukocytes and glandular epithelium, during the pre-implantation phase of pregnancy.     

Leukemia inhibitory factor ligand-receptor signaling is important for uterine receptivity and implantation in golden hamsters (Mesocricetus auratus)

Blastocyst implantation occurs in the progesterone-primed uterus of hamsters, but not in mice where the progesterone-primed uterus requires estrogen influence. Leukemia inhibitory factor (Lif), an estrogen-regulated gene in mice, is an absolutely needed cytokine for uterine receptivity and implantation in this species. This study aimed to evaluate the importance of Lif ligand-receptor signaling during uterine receptivity and implantation in hamsters. We investigated whether or not the uterine expression patterns of Lif and its receptors, Lif-r and gp130, during the periimplantation period of pregnancy and its hormonal regulation in the ovariectomized hamster correlate with some of the vital phases of uterine changes during early pregnancy. Uterine Lif, Lif-r, and gp130 mRNA expressions were examined by Northern and in situ hybridization. During the uterine preparatory phase for implantation, Lif, Lif-r, and gp130 were expressed either in the gland, luminal epithelium or both. As the implantation process began, Lif expression was minimal, but Lif-r and gp130 extended to the decidual areas. This decidual expression of Lif-r and gp130 was not dependent on the presence of the embryo since these genes were expressed in the suture-induced deciduomata. We also observed that, while the uterine Lif was induced by estrogen, Lif-r and gp130 were induced by progesterone in ovariectomized hamsters. Additionally, we show that a Lif antibody when instilled intraluminally on day 3 of pregnancy reduced the number of implantation sites. Taken together, these data suggest that Lif signaling is important for uterine receptivity and implantation in hamsters.     

Eupatorium japonicum extract regulates inflammation through suppression of the TRIF-dependent signaling pathway of toll-like receptors

Inflammation can be mediated by invading microbial pathogens. Toll-like receptors (TLRs) recognize invading microbial pathogens, inducing innate immune responses. Broadly, the activation of TLRs induces two major downstream signaling pathways, myeloid differential factor 88 (MyD88)- and Toll/interleukin 1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways, which lead to the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3). To evaluate the therapeutic potential of the ethanol extracts of flowers of Eupatorium japonicum Thunb (EJE), its effect on signal transduction via the TLR signaling pathways induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly[I:C]) was examined. EJE suppressed the activation of NF-κB and IRF3 induced by LPS or poly[I:C]. EJE also inhibited LPS- or poly[I:C]-induced IRF3 phosphorylation as well as interferon-inducible genes, such as interferon inducible protein-10. These results suggest that EJE can modulate TLR signaling pathways, realizing effective therapeutic options for chronic inflammatory diseases.     

Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway

The nutritional and endocrine factors affecting protein translation in the bovine mammary gland, and the molecular mechanisms mediating their effects, are not well understood. The objective of this study was to assess the role of the mammalian target of rapamycin (mTOR) signaling pathway in the regulation of mammary protein synthesis by nutrients and hormones. Mammary epithelial acini isolated from lactating dairy cows were treated in medium containing, alone or in combination, a mixture of AA or glucose and acetate (GA) as energy substrates, or a combination of the lactogenic hormones hydrocortisone, insulin, and prolactin (HIP). Changes in the rate of mammary protein synthesis and the phosphorylation state of components of the mTOR signaling pathway were measured. Mammary protein synthesis was 50% higher with increased availability of AA in medium. The presence of GA or treatment of mammary acini with HIP alone did not affect mammary protein synthesis. The stimulation of mammary protein synthesis by AA was enhanced by HIP treatment, but not by the presence of GA in medium. Treatment of mammary acini with HIP induced the phosphorylation of protein kinase B. This effect was augmented in the presence of either AA or GA in medium. The stimulation of mammary protein synthesis by AA and its enhancement by HIP were associated with increased phosphorylation of the mTOR substrates, p70 ribosomal protein S6 kinase-1, and eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1), and dissociation of 4E-BP1 from eIF4E. The results suggest that nutrients and hormones may modulate mammary protein synthesis through the mTOR signaling pathway.     

Combination of mineral trioxide aggregate and propolis promotes odontoblastic differentiation of human dental pulp stem cells through ERK signaling pathway

Food Science and Biotechnology - The aim of this study is to investigate combined effects of mineral trioxide aggregate (MTA) and propolis on odontoblastic differentiation of human dental pulp stem...     

Acetate regulates milk fat synthesis through the mammalian target of rapamycin/eukaryotic initiation factor 4E signaling pathway in bovine mammary epithelial cells

Acetate is a short-chain fatty acid (SFA) that is the major substrate for de novo fatty acid synthesis. The mammalian target of rapamycin/eukaryotic initiation factor 4E (mTOR/eIF4E) signaling pathway is involved in fat synthesis. However, the effect and mechanism of acetate on fatty acid synthesis by the mTOR/eIF4E signaling pathway is unclear in bovine mammary epithelial cells (BMECs). The objectives of this study were to investigate the effect of acetate on cell viability, triacylglycerol (TG), and mRNA expression of the genes related to lipid synthesis. The mechanism of acetate regulation milk fat synthesis through the mTOR/eIF4E signaling pathway was assessed by blocking the mTOR signaling pathway and silencing eIF4E in BMECs. Third-passage BMECs were allocated to 6 treatments including 0, 4, 6, 8, 10, and 12 mM acetate to evaluate the effect of acetate on lipid synthesis; the optimum concentration in the first study was selected for the subsequent study. Subsequently, cells were randomly allocated to 4 treatments, 1 control group and 3 treated groups, consisting of acetate (6 mM), rapamycin (100 nM), and acetate + rapamycin to test the role of mTOR signaling pathway response to acetate in milk lipid synthesis. Finally, eIF4E was silenced by small interfering RNA (siRNA) to detect the role of eIF4E in milk lipid synthesis. Treatments included control, eIF4E siRNA, acetate (6 mM), and acetate+ eIF4E siRNA. Results showed that acetate increased TG accumulation and the relative expression of fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACACA), fatty acid-binding protein 3 (FABP3), sterol regulatory element binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPARG), mTOR, eIF4E, P70 ribosomal protein S6 kinase-1 (S6K1), and 4E-binding protein-1 (4EBP1) in a dose-dependent manner. Rapamycin effectively inhibited the positive effect of acetate on the relative expression of mTOR, eIF4E, S6K1, 4EBP1, FASN, ACACA, FABP3, stearoyl-CoA desaturase (SCD1), SREBP1, and PPARG. The upregulation of acetate on the relative expressions of FASN, ACACA, SCD1, and SREBP1 was suppressed when eIF4E was knocked down. It suggested that acetate regulated milk fat synthesis through mTOR/eIF4E signaling pathway in BMECs.     

Genes of the transforming growth factor-beta signalling pathway are associated with pre-implantation embryonic development in cattle

One of the main factors affecting cattle fertility is pre-implantation development of the bovine embryo, which is a complex process regulated by various signal-transduction pathways. The transforming growth factor-β (TGF-β) signalling system, which is responsible for many biological processes including cell proliferation, differentiation and apoptosis, also is involved in embryo development. We hypothesized that altered expression of TGF-β genes in pre-implantation bovine embryos is associated with morphological abnormalities of these embryos. To test this hypothesis, we produced embryos in vitro and classified them at the blastocyst stage as either normally developed blastocysts or degenerates (growth-arrested embryos). The expression patterns of 25 genes from the TGF-β pathway were assessed using quantitative real time PCR. Ten genes showed differential expression between the two embryo groups, four genes displayed similar expressional profiles, and 11 genes had no detectable expression. An altered expression profile was statistically significant for 10 of the 14 expressed genes, and all were up-regulated in degenerate embryos vs. blastocysts. Furthermore, genomic association analysis of the cows from which embryos were produced revealed a significant association of ID3 and BMP4 polymorphisms--two of the most significant differentially expressed genes--with fertilization rate and blastocyst rate, respectively. Taken together, we conclude that TGF-β pathway genes, especially BMP4 and ID3 play a vital function in the regulation of pre-implantation embryo development at both embryo and maternal levels. Hence, these genes may be suitable as genetic markers for embryo development and fertility in cattle.     

Kefir peptides exhibit antidepressant-like activity in mice through the BDNF/TrkB pathway

Depression is a prevalent, stress-related mental disorder that can lead to serious psychiatric diseases with morbidity and high mortality. Although some functional fermented dairy drinks have promising anxiolytic and antidepressant effects, the mechanism is still not clear. To determine the antidepressant-like effect and the potential molecule mechanism of kefir peptides (KP), various behavioral tests, including the elevated plus maze test, open field test, forced swimming test, and tail suspension test, were used. Administration of 150 mg/kg KP in mice reduced the duration of immobility in the forced swimming test and tail suspension test, elevated the time spent in the open arm and center zone in the elevated plus maze test, and increased the total distance traveled, average speed, and time spent in the center zone in the open field test compared with the mock group. These results indicated that KP dramatically ameliorated the depression-like behaviors. Kefir peptides were further isolated and identified using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry, from which 3 peptides were identified and designated KFP-1, KFP-3, and KFP-5. Among these peptides, administration of KFP-3 (15 AA residues) remarkably decreased immobility time in the forced swimming test and increased mobility time in the tail suspension test. Therefore, KFP-3 may be the major active peptide with antidepressant activity in KP. Overexpression of brain-derived neurotrophic factor, phosphorylated tropomyosin receptor kinase B, and phosphorylated ERK1/2 protein levels could be detected in the hippocampus under KP administration. Therefore, we suggest that KP improves depressive-like behaviors by activating the brain-derived neurotrophic factor–phosphorylated tropomyosin receptor kinase B signaling pathway. Kefir peptides may serve as a new type of antidepressant dairy product and may provide potent antidepressant effects for clinical use.     

Reduced embryonic survival in rainbow trout resulting from paternal exposure to the environmental estrogen 17alpha-ethynylestradiol during late sexual maturation

Exposure of fishes to environmental estrogens is known to affect sexual development and spawning, but little information exists regarding effects on gametes. This study evaluated embryonic survival of offspring from male rainbow trout (Oncorhynchus mykiss) exposed to 17alpha-ethynylestradiol (EE(2)) using an in vitro fertilization protocol. Males were exposed at either 1800 or 6700 degree days ( degrees d) (i.e. 161 or 587 days post-fertilization (dpf)) to test for effects on testes linked to reproductive ontogeny. At 1800 degrees d, fish were beginning testicular differentiation and were exposed to 109 ng EE(2)/l for 21 days. At 6700 degrees d, fish have testes containing spermatocytes and spermatids and were exposed for 56 days to either 0.8, 8.3, or 65 ng EE(2)/l. Semen was collected at full sexual maturity in each group and used to fertilize eggs pooled from several non-exposed females. Significant decreases in embryonic survival were observed only with the 6700 degrees d exposure. In 0.8 and 8.3 ng EE(2)/l treatments, embryo survival was significantly reduced at 19 dpf when compared with the control. In contrast, an immediate decrease in embryonic survival at 0.5 dpf was observed in the 65 ng EE(2)/l treatment. Blood samples collected at spawning from 6700 degrees d exposed males revealed a significant decrease in 11-ketotestosterone and a significant increase in luteinizing hormone levels for the 65 ng EE(2)/l treatment when compared with the other treatment groups. Results indicate that sexually maturing male rainbow trout are susceptible to EE(2) exposure with these fish exhibiting two possible mechanisms of reduced embryonic survival through sperm varying dependant on EE(2) exposure concentrations experienced.     

Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway

Ovarian cancer is a significant malignancy for women in the western world, and its death rate has remained unchanged over the past 50 years, leaving room for proper chemoprevention. Kaempferol is a natural flavonoid widely distributed in fruits and vegetables, and epidemiological studies have found a negative correlation between kaempferol consumption and ovarian cancer risk. To understand the mechanism behind this negative correlation, we investigated kaempferol's ability to induce apoptosis in A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines. Kaempferol inhibited cell proliferation but did not cause necrosis in all 3 cell lines. For the apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating an intrinsic apoptosis pathway. Western blot analysis revealed that protein levels of Bcl-x(L) were decreased in ovarian cancer cells, while p53, Bad, and Bax proteins were up-regulated by kaempferol treatment. Our data indicate that kaempferol induces apoptosis in ovarian cancer cells through regulating pro-apoptotic and anti-apoptotic protein expressions in the intrinsic apoptosis pathways, and is a good candidate for the chemoprevention of ovarian cancers in humans. Further studies in animal models and clinical trials are therefore warranted.     

猴头菇麦饭石母子酱油的营养和功能性探讨

在传统母子酱油生产过程中分别添加麦饭石、猴头菇和甘草等材料,在此基础上生产的猴头菇麦饭石母子酱油既保持了其传统特色。又增加了营养保健功能。     

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.     

Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101

Sourdough lactic acid bacteria (LAB) need to be adapted to a highly acidic and, therefore, challenging environment. Different mechanisms are employed to enhance competitiveness, among which conversion of arginine into ornithine through the arginine deiminase (ADI) pathway is an important one. A combined molecular and kinetic approach of the ADI pathway in Lactobacillus fermentum IMDO 130101, a highly competitive sourdough LAB strain, identified mechanisms with advantageous technological effects and quantified the impact of these effects. First, molecular analysis of the arcBCAD operon of 4.8 kb revealed the genes encoding the enzymes ornithine transcarbamoylase, carbamate kinase, arginine deiminase, and an arginine/ornithine (A/O) antiporter, respectively, with an additional A/O antiporter 702.5 kb downstream of the ADI operon. The latter could play a role in citrulline transport. Second, pH-controlled batch fermentations were carried out, generating data for the development of a mathematical model to describe the temporal evolution of the three amino acids involved in the ADI pathway (arginine, citrulline, and ornithine) as a result of the activity of these enzymes and transporter(s). Free arginine in the medium was converted completely into a mixture of citrulline and ornithine under all conditions tested. However, the ratio between these end-products and the pattern of their formation showed variation as a function of environmental pH. Under optimal pH conditions for growth, citrulline release and some further conversion into ornithine was observed. When growing under sub-optimal pH conditions, ornithine was the main product of the ADI pathway. These kinetic data suggest a role in adaptation of L. fermentum IMDO 130101 to growth under sub-optimal conditions.     

Anti-cancer activity of the ethylacetate fraction from Orostachys japonicus for modulation of the signaling pathway in HepG2 human hepatoma cells

The molecular mechanisms of the ethylacetate (EtOAc) fraction from Orostachys japonicus (OJE) (including gallic acid, kaempferol, and quercetin) for anti-cancer activities in HepG2 cells were investigated. Apoptosis was detected using morphological observation of nuclear changes and investigation of phosphatidylserine exposure at the cytoplasmic membrane using FITC-Annexin V/PI staining. Activities of the apoptotic factors bcl-2, bax, cytochrome c, pro-caspase-3, 8, and 9, as well as MAPKs levels, were measured using western blotting. Some morphologic features of apoptosis were identified using confocal microscopy. OJE caused the expression of apoptotic proteins to change, as evidenced by an increased bax/bcl-2 ratio and increased expression of cytochrome c, and decreased expressions of pro-caspase-3, 8, and 9. After HepG2 cells were exposed to OJE, expressions of p-JNK and p-ERK1/2 increased in a dose dependent manner. OJE exhibits anti-cancer activity via apoptosis induction through a mitochondria dependent signaling pathway in HepG2 cells.     

Degradation pathway of carnosic acid in methanol solution through isolation and structural identification of its degradation products

Carnosic acid is the primary rosemary-derived phenolic diterpenes with greatest antioxidant activity, but it is rather unstable in methanol solution. Understanding the degradation pathway of carnosic acid and the interaction between these phenolic compounds is important if they are to be utilized to a greatest effect. In this study, the HPLC analysis of an aged methanolic solution of carnosic acid revealed that carnosic acid was degraded, and several compounds were produced in the solution. From the time-course and quantitative analyses of the formation of the products and their structural analysis, a novel degradation pathway of carnosic acid in methanol solution is proposed. Carnosic acid can firstly decompose into carnosic acid quinone in methanol, which was the first time to be confirmed the existence of carnosic acid quinone. Carnosic acid quinone is the quinone intermediate of carnosic acid that can be reduced into carnosic acid or can be further converted into carnosol. Carnosol can further decompose to form epirosmanol, rosmanol, 7-methoxyrosmanol, and 7-methoxy-epirosmanol. The latter three compounds were identified as the final degradation products of carnosic acid in methanol because they appeared rather stable in the study.     

A mathematical model of the interaction between bovine blastocyst developmental stage and progesterone-stimulated uterine factors on differential embryonic development observed on Day 15 of gestation

A complex interaction between the developing bovine embryo and the growth potential of the uterine milieu it inhabits results in an embryo capable of developing past the maternal recognition stage and on to a successful pregnancy. Previously, we observed variation in the lengths of embryos recovered 8 d after bulk transfer of Day 7 in vitro-produced (IVP) blastocysts into the same uterus. Potential causes of the differential embryonic growth were examined and modeled using 2 rounds of bulk (n = 4–6) IVP transfers and recovery of these embryos 8 d later. Morphological and gene expression measurements of the embryos were determined and the progesterone concentration of the cows was measured throughout the reproductive cycle as a reflection of the status of the uterine environment. These data were used to develop and evaluate a model that describes the interaction between the uterine environment and the growth rate of the developing embryo. Expression of 6 trophectoderm genes (IFNT, TKDP1, PAG11, PTGS2, DKK1, and PDPN) was correlated with conceptus length. The model determined that if the embryo develops to blastocyst stage, the uterine environment, driven by progesterone, is a more important component than blastocyst size in the stimulation of embryonic growth rate to ensure adequate interferon tau (IFNT) for pregnancy recognition. We detected an effect of Day 7 progesterone on the expression of all 6 genes, embryonic disc size, and trophectoderm length on Day 15. We also found effects of embryo transfer size on trophectoderm length and expression of IFNT and PAG11 on Day 15. Lower energy balance over the period from transfer to recovery was associated with reduced embryo growth to Day 15, and this effect was independent of progesterone. Energy balance also affected expression of PDPN and TKDP1 on Day 15. We observed an effect of energy balance from transfer to recovery on embryo survival in cows with partial embryo losses, where embryo factors dominate embryo survival, with cows with greater energy balance having lower embryo losses. This effect was independent of energy balance 40 d before transfer and suggests that energy balance has direct, immediate effects on the embryo and maternal environment during this period. Furthermore, energy balance effects on embryo survival in cows with partial embryo losses were largely mediated by expression of TKDP1, PAG11, and PDPN. These results provide candidate signaling pathways for the effect of progesterone and energy balance on embryo growth and survival.