Interference of pseudorabies virus infection on functions of porcine granulosa cells via apoptosis modulated by MAPK signaling pathways.

BACKGROUND: Pseudorabies virus (PRV) is one of the major viral pathogens leading to reproductive disorders in swine. However, little is known about the effects of PRV infection on porcine reproductive system. Ovarian granulosa cells are somatic cells surrounding oocytes in ovary and required for folliculogenesis. The present study aimed to investigate the interference of PRV on functions of porcine ovarian granulosa cells in vitro. METHODS: Primary granulosa cells were isolated from porcine ovaries. To investigate the PRV infectivity, transmission electron microscopy (TEM) was used to check the presence of viral particles, and the expression of viral gE gene was detected by quantitative real-time PCR (qPCR) in PRV-inoculated cells. After PRV infection, cell viability was detected by MTS assay, Ki67 for proliferative status was determined by immunofluorescence assay (IFA), cell cycle and apoptosis were detected by flow cytometry, and progesterone (P4) and estradiol (E2) were determined by radioimmunoassay. The checkpoint genes of cell cycle and apoptosis-related proteins were studied by qPCR and western blotting. RESULTS: Virus particles were observed in the nucleus and cytoplasm of PRV-infected granulosa cells by TEM imaging, and the expression of viral gE gene increased in a time-dependent manner post infection. PRV infection inhibited cell viability and blocked cell cycle at S phase in porcine granulosa cells, accompanied by decreases in expression of Ki67 protein and checkpoint genes related to S phase. Radioimmunoassay revealed decreased levels in P4 and E2, and the expressions of key steroidogenic enzymes were also down-regulated post PRV-infection. In addition, PRV induced apoptosis with an increase in Bax expression and activation of caspase 9, and the phosphorylation of JNK, ERK and p38 MAPKs were significantly up-regulated in porcine ovarian granulosa cells post PRV infection. CONCLUSIONS: The data indicate that PRV causes infection on porcine ovarian granulosa cells and interferes the cell functions through apoptosis, and the MAPK signaling pathway is involved in the viral pathogenesis.

Trophoblast-specific knockdown of CSPG4 expression causes pregnancy complications with poor placentation in mice.

The multifunctional molecule chondroitin sulfate proteoglycan 4 (CSPG4/NG2) plays key roles in organogenesis and tumorigenesis. However, its roles in placentation remain unclear. In this study, CSPG4 expression in human and mouse placentas was investigated through immunohistochemistry (IHC), qPCR and western blotting. The theoretical structure and function of CSPG4 were assessed using bioinformatic tools, and the functions of CSPG4 in fetal and placental development were investigated using a mouse model established by trophoblast-specific CSPG4 knockdown and a trophoblast cell line with CSPG4 knockout by lentivirus infection. The results showed that CSPG4 was mainly located in trophoblasts in both human placentas and mouse placentas, with a higher level in preeclampsia (PE) placentas than in healthy control placentas. Furthermore, there was a trend of increasing expression in mouse placentas during pregnancy. The 3D structure of CSPG4 was visualized using an M model composed of two chains, and the structure implied that CSPG4 was a multifunctional molecule containing multiple pockets with multiligand binding sites and enzyme active sites. Trophoblast-specific CSPG4 knockdown caused frequent fetal loss, and viable fetal development was restricted by poor placentation, with mice placentas having reduced weight and width. The proliferation and invasion of CSPG4-knockout trophoblasts were significantly inhibited, and as such, the molecular signaling of AKT and ERK phosphorylation was inhibited, and the expression of MMP2 and MMP9 was reduced. In summary, CSPG4 deficiency inhibited trophoblast proliferation and invasion, which was associated with AKT, ERK and MMP signaling. CSPG4 deficiency also caused pregnancy complications with poor placentation in mice.

α-Solanine Inhibits Proliferation, Invasion, and Migration, and Induces Apoptosis in Human Choriocarcinoma JEG-3 Cells In Vitro and In Vivo.

α-Solanine, a bioactive compound mainly found in potato, exhibits anti-cancer activity towards multiple cancer cells. However, its effects on human choriocarcinoma have not been evaluated. In the present study, we investigated the effect of α-solanine on cell proliferation and apoptosis in human choriocarcinoma in vitro and in vivo. The results showed that α-solanine, at concentrations of 30 µM or below, did not affect the cell viability of the choriocarcinoma cell line JEG-3. However, colony formation was significantly decreased and cell apoptosis was increased in response to 30 µM α-solanine. In addition, α-solanine (30 µM) reduced the migration and invasion abilities of JEG-3 cells, which was associated with a downregulation of matrix metalloproteinases (MMP)-2/9. The in vivo findings provided further evidence of the inhibition of α-solanine on choriocarcinoma tumor growth. α-Solanine suppressed the xenograft tumor growth of JEG-3 cells, resulting in smaller tumor volumes and lower tumor weights. Apoptosis was promoted in xenograft tumors of α-solanine-treated mice. Moreover, α-solanine downregulated proliferative cellular nuclear antigen (PCNA) and Bcl-2 levels and promoted the expression of Bax. Collectively, α-solanine inhibits the growth, migration, and invasion of human JEG-3 choriocarcinoma cells, which may be associated with the induction of apoptosis.

α-Solanine Causes Cellular Dysfunction of Human Trophoblast Cells via Apoptosis and Autophagy.

The trophoblast, an embryonic tissue, exerts a crucial role in the processes of implantation and placentation. Toxins in food can cause malfunction of trophoblasts, resulting in apoptosis, oxidative stress, and abnormal angiogenesis. α-solanine, a steroidal glycoalkaloid, has antitumor properties on several cancer cells. However, its effect on human trophoblasts has not been elucidated. In this study, human extravillous trophoblast HTR-8/SVneo cells were exposed to α-solanine. Cellular functions including proliferation, migration, invasion, tube formation, and apoptosis were assessed. To monitor autophagic flux, trophoblasts were transfected with a mCherry-GFP-LC3B vector using lentiviral transduction, and expression of autophagy-related biomarkers including Beclin 1, Atgl3, and microtubule-associated protein 1 light chain-3 (MAP1-LC3) were detected. The results show that application of 20 µM α-solanine or above inhibited the cell viability, migration, invasion, and tube formation of the human trophoblast. Cell cycle was arrested at S and G2/M phases in response to 30 µM α-solanine. α-solanine induced apoptosis of HTR-8/SVneo cells and triggered autophagy by increasing the autophagic gene expression and stimulating the formation of autophagosome and autophagic flux. In conclusion, α-solanine can impair the functions of human trophoblast cells via activation of cell apoptosis and autophagy.

Spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacenta for regulating of placental angiogenesis through VEGF-mediated signalling.

Non-infectious prenatal mortality severely affects the porcine industry, with pathological placentation as a likely key reason. Previous studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) deficiency causes defects in the uteroplacental vasculature and induces embryonic losses in mice. However, its role in porcine placental angiogenesis remains unclear. In the present study, PPARγ expression was investigated in porcine uteroplacental tissues at gestational day (GD) 25, GD40 and GD70 via quantitative polymerase chain reaction (qPCR), Western blot and immunohistochemistry (IHC). Moreover, the roles of PPARγ in porcine placental angiogenesis were investigated using a cell model of porcine umbilical vein endothelial cells (PUVECs) to conduct proliferation, migration and tube formation assays in vitro and a mouse xenograft model to assess capillary formation in vivo. The results showed that PPARγ was mainly located in the glandular epithelium, trophoblast, amniotic chorion epithelium and vascular endothelium, as indicated by the higher expression levels at GD25 and GD40 than at GD70 in endometrium and by higher expression levels at GD40 and GD70 than at GD25 in placenta. Moreover, PPARγ expression was significantly downregulated in placenta with dead foetus. In PUVECs, knocking out PPARγ significantly inhibited proliferation, migration and tube formation in vitro and inhibited capillary formation in mouse xenografts in vivo by blocking S-phase, promoting apoptosis and downregulating the angiogenic factors of VEGF and its receptors. Overall, the spatiotemporal heterogeneity of PPARγ expression in porcine uteroplacental tissue suggests its vital role in endometrial remodelling and placental angiogenesis, and PPARγ regulates placental angiogenesis through VEGF-mediated signalling.

R-spondin3 promotes the tumor growth of choriocarcinoma JEG-3 cells.

R-spondin3 (RSPO3), an activator of Wnt/ß-catenin signaling, plays a key role in tumorigenesis of various cancers, but its role in choriocarcinoma remains unknown. To investigate the effect of RSPO3 on the tumor growth of choriocarcinoma JEG-3 cells, the expression of RSPO3 in human term placenta was detected, and a stable RSPO3-overexpressing JEG-3 cell line was established via lentivirus-mediated transduction. The expression of biomarkers involved in tumorigenicity was detected in the RSPO3-overexpressing JEG-3 cells, and cell proliferation, invasion, migration, and apoptosis were investigated. Moreover, soft agar clonogenic assays and xenograft tumorigenicity assays were performed to assess the effect of RSPO3 on tumor growth in vitro and in vivo. The results showed that RSPO3 was widely expressed in human term placenta and overexpression of RSPO3 promoted the proliferation and inhibited the migration, invasion, and apoptosis of the JEG-3 cells. Meanwhile, RSPO3 overexpression promoted tumor growth both in vivo and in vitro. Further investigation showed that the phosphorylation levels of Akt, phosphatidylinositol 3-kinase (PI3K), and ERK as well the expression of ß-catenin and proliferating cell nuclear antigen (PCNA) were increased in the RSPO3-overexpressing JEG-3 cells and tumor xenograft. Taken together, these data indicate that RSPO3 promotes the tumor growth of choriocarcinoma via Akt/PI3K/ERK signaling, which supports RSPO3 as an oncogenic driver promoting the progression of choriocarcinoma.

Molecular Detection and Genotyping of Toxoplasma gondii in Pigs for Human Consumption in Hunan Province, China.

Toxoplasma gondii is one of the most important sources of foodborne diseases. In this study, the molecular prevalence and genotypes of T. gondii were investigated in pigs in Hunan province, China. A total of 339 brain tissue samples of pigs were collected from April 2015 to December 2017 in Hunan province and were used to detect the T. gondii B1 gene. Of these, 34 (10%; 95% confidence interval: 8.7-12.6) samples were tested positive for the T. gondii B1 gene. Positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5' + 3' SAG2, alter. SAG2], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using polymerase chain reaction-restriction fragment length polymorphism technology. Moreover, one sample was identified as genotype ToxoDB#10 (Type I), and another sample was suspected to be unusual genotype ToxoDB#61 that has never been reported in China. This study showed that T. gondii is prevalent in pigs in Hunan province, posing a food safety threat to the public health in the investigated areas. Our result has implications for better understanding the genetic diversity of T. gondii infections in animals in China.

Peroxisome proliferator­activated receptor γ mediates porcine placental angiogenesis through hypoxia inducible factor­, vascular endothelial growth factor­ and angiopoietin­mediated signaling.

Peroxisome proliferator-activated receptor (PPAR) γ has been reported to be implicated in placentation in mice. Previous studies have demonstrated that PPARγ is also expressed in porcine placenta, primarily localized in vascular endothelial cells (VECs). The present study aimed to investigate the roles of PPARγ during porcine placental angiogenesis and examine the molecular mechanisms involved in its actions. VECs were incubated with the PPARγ agonist rosiglitazone and the antagonist T0070907, and their angiogenic potential was evaluated using cellular impedance, wound healing and tube formation assays. Reverse transcription­quantitative polymerase chain reaction was used to assess the mRNA expression levels of angiogenic factors, including hypoxia­inducible factors (HIFs), vascular endothelial growth factor (VEGF) isoforms, VEGF receptors (VEGFRs) and angiopoietins (Angs). The results demonstrated that the adhesive, proliferative and migratory capabilities of VECs were potentiated by rosiglitazone and suppressed by T0070907. Notably, tube formation was invariably promoted during PPARγ activation and blockade. The mRNA expression levels of HIF1α, HIF2α, VEGFR2, VEGF188 and Ang­1 were revealed to be upregulated following treatment of VECs with rosiglitazone, whereas they were downregulated following treatment with T0070907. However, the mRNA expression levels of placental growth factor and VEGF120 were consistently downregulated following PPARγ activation and blockade, whereas VEGF164 mRNA levels remained unaltered. The results of the present study suggested that PPARγ may mediate porcine placental angiogenesis, by interfering with HIF­, VEGF­ and angiopoietin­mediated signaling pathways.

Dietary Supplementation of Astragalus Polysaccharides Enhanced Immune Components and Growth Factors EGF and IGF-1 in Sow Colostrum.

Colostrum is the main external resource providing piglets with nutrients and maternal immune molecules. Astragalus polysaccharides (APS) have been used as immunopotentiators in vitro and several animal models. This study aimed to determine the effects of APS on immune factors in sow colostrum and milk. The sow diet was supplemented with APS one week before the expected delivery date. Colostrum and milk were collected and designated as 0 h- (onset of parturition), 12 h-, and 24 h-colostrum and 36 h-milk postpartum. Samples were measured using porcine immunoglobulin (Ig) G, IgM, classical swine fever virus antibody (CSFV Ab), epidermal growth factor (EGF), and insulin-like growth factor- (IGF-) 1 ELISA Quantitation Kits. Dietary supplementation of APS significantly enhanced the presence of IgG, IgM, EGF, and IGF-1 in 0 h-colostrum (P < 0.001). The blocking rates of CSFV Ab were increased in samples from APS-supplemented sow when compared to those from the matched samples without APS treatment. The results indicate that supplement of APS could improve the immune components in sow colostrum and/or milk; and status of some specific vaccination could be determined through using colostrum or early milk in sow.

Sexing murine embryos with an indirect immunofluorescence assay using phage antibody B9-Fab against SDM antigen.

The use of serologically detectable male (SDM; also called H-Y) antigens to identify male embryos may be limited by the source of anti-SDM antibody. In the present study, novel anti-SDM B9-Fab recombinant clones (obtained by chain shuffling of an A8 original clone) were used to detect SDM antigens on murine embryos. Murine morulae and blastocysts (n=138) were flushed from the oviducts of Kunming mice and incubated with anti-SDM B9-Fab for 30 min at 37°C. With an indirect immunofluorescence assay, the membrane and inner cell mass had bright green fluorescence (presumptive males). Overall, 43.5% (60/138) were classified as presumptive males and 56.5% (78/138) as presumptive females, with 85.0 and 88.5% of these, respectively, confirmed as correct predictions (based on PCR analysis of a male-specific [Sry] sequence). We concluded that the anti-SDM B9-Fab molecule had potential for non-invasive, technically simple immunological sexing of mammalian embryos.

Engineering the male-specificity of Fab against SDM antigen by chain shuffling.

High-titer serologically detected male (SDM) antibody fragments are essential for specific binding to the SDM antigen and promoting its application. The A8 clone previously obtained from an original phage antibody library was further affinity-matured by light- and high-chain shuffling respectively, to generate the end product B9 clone. The binding capacity of B9 phage Fabs to male splenocytes doubled the value of its parental A8 clone (determined using ELISA). Based on immunofluorescent staining, B9-Fabs mainly bound to the surface antigen of male splenocytes and recognized testicular cells. The resulting B9-Fabs detected a single protein (approximately 40 kDa determined using Western blot analysis of male splenocytes and testis); its high SDM antigen binding ability might have been because of mutation sites and varied lengths of the amino acid sequences in the complementarity determining regions-3 of the κ and Fd chains. The new recombinant clones of Fab that were phage-enhanced using chain shuffling were candidate molecules for investigating molecular mechanisms of SDM antigens specific binding and applications.

Viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b.

The viral distribution and lesions in Kunming mice experimentally infected with porcine circovirus type 2b (PCV-2b) were investigated. Seventy special pathogen free mice were divided into 2 groups with 35 mice in each group. The test group (TG) was infected with PCV-2b, the control group (CG) was inoculated with sterile cell cultures. Five mice in each group were sacrificed at 3, 7, 14, 21, 28, 35 and 42 dpi (day post infection), respectively. Necropsies were performed on all mice and tissues were collected for testing by histopathology, immunohistochemistry, transmission electron microscope (TEM) and polymerase chain reaction (PCR). Apoptosis and necrosis in lymphoid organs were observed in virus-infected mice, and became severe from 14 to 28 dpi. The proportion of PCV-2b antigen-positive cells was moderate in lung, heart, thymus, liver or kidney, and low in brain from TG. In spleen and cervical lymph node, the proportions of PCV-2b antigen-positive cells were low to high from 7 to 28 dpi, and moderate from 35 to 42 dpi. PCV-2b DNA was detected in all tissues examined in TG from 7 to 42 dpi. Viral inclusion bodies presented in the cytoplasm of lymphocytes, macrophages, hepatocytes, podocytes, neurocytes, spermatids and uterine epithelial cells in TG. In CG, no viruses and viral lesions were detected. PCV-2b could replicate in mice, and PCV-2b associated lesions in mice were similar to those observed in pigs. The present results indicate that it is possible to use Kunming mouse as an animal model for PMWS research.

Identification of mouse 8-cell embryo stage-specific genes by Digital Differential Display.

Preimplantation development is critical for successful implantation and pregnancy. In the mouse preimplantation embryo, the first event of morphological and cellular differentiation is established during polarization and compaction at the 8-cell stage. The considerable cell surface and cytoplasmic changes and formation of different populations of cells at the 8-cell stage are fundamentally important for the development of all organisms. To determine genes that are specifically expressed at this crucial stage of embryo development and also to shed light on the different mechanisms that could be of importance during embryo development, we investigated mouse 8-cell and 4-cell embryo stage-specific genes using Digital Differential Display (DDD). The 8-cell stage-specific genes were sorted according to their ontology data from the Database for Annotation, Visualization and Integrated Discovery (DAVID), which outlines possible roles for the genes expressed at the 8-cell stage. This study highlights how online tools can be used to identify genes involved in embryo development. Identification of the 8-cell embryo stage-specific genes would open new opportunities for understanding molecular networks during the mid-preimplantation gene activation. Using bioinformatic tools, such as Digital Differential Display and DAVID, it will be possible to identify genes expressed at the 8-cell stage that are likely to be involved in mammalian preimplantation embryo development. Our results may provide a new foundation for molecular control at the onset of embryonic development in mammals, and should be of interest to the scientific community.

Construction and characterization of phage display library: recognition of mouse serologically detected male (SDM) antigen.

Improvement of animal embryo sexing depends upon high-titer serologically detected male (SDM) antibody fragments. SDM sera collected from isogenic C57BL/7 female mice after inoculation with male spleen cells were characterized and used for construction of a recombinant Fab antibody library against SDM antigen, and used for analysis of the binding capacity and specificity to SDM antigen. The heavy-chain Fd and full-length light-chain kappa were amplified by RT-PCR from a mouse (#6) that'ed high-titer antiserum. The amplified product was inserted into the pComb3 vector followed by co-infections with the help phage VCSM 13 for construction of the phage library, which gave 1.5x10(7) colonies with the titer of 3.2x10(11) pfu/ml by a recombination rate of 80%. Sequence analysis of the PCR products of plasmid DNA of E5 clones showed that V(H) and V(kappa) had common characteristics shared by other known variable region of antibodies. The Fab antibody libraries against SDM antigen were enriched by three cycles of affinity enrichment with male spleen cells, and two cycles of non-specific absorption with female spleen cells. The ELISA results showed that 9 of 15 clones had binding capacity to the SDM antigen. This is the first report on a phage display library of SDM antigen. The mouse Fab antibody library could be used for identifying SDM antigen, and for the development of sex determination of early embryos in mammals.

[Cloning and analysis of phage Fab antibodies of mouse male specific antigen].

To clone mouse phage antibodies against H-Y antigen from a phage antibody library, three cycles of affinity enrichment of the mouse phage antibody library with male spleen cells and two cycles of nonspecific absorption with female spleen cells were performed. The presence of mouse Fab on the phage surface was determined by ELISA and sequence analysis. 9 of 15 strains can bind to male spleen cells with the specific activity. Recombination rate of the phage antibody library clones is 60%. Sequence analysis of the PCR products of plasmid DNA of E5 clones show VH and Vkappa had common characteristics shared by other known variable region of antibodies. The mouse phage Fab antibody could be used for identifying H-Y antigen, and for the development of sex determination of early embryos in mammals.