PLAC8, a new marker for human interstitial extravillous trophoblast cells, promotes their invasion and migration.

Proper differentiation of trophoblast cells in the human placenta is a prerequisite for a successful pregnancy, and dysregulation of this process may lead to malignant pregnancy outcomes, such as preeclampsia. Finding specific markers for different types of trophoblast cells is essential for understanding trophoblast differentiation. Here, we report that placenta-specific protein 8 (PLAC8) is specifically expressed in the interstitial extravillous trophoblast cells (iEVTs) on the fetomaternal interface. Using model systems, including placental villi-decidua co-culture, iEVTs induction by using primary trophoblast cells or explants, etc., we found that PLAC8 promotes invasion and migration of iEVTs. Mechanistically, time-lapse imaging, GTPase activity assay, co-immunoprecipitation and RNA-seq studies show that PLAC8 increases the Cdc42 and Rac1 activities, and further induces the formation of filopodia at the leading edge of the migratory trophoblast cells. More interestingly, PLAC8 is significantly upregulated under hypoxia and expression of PLAC8 is higher in iEVTs from preeclamptic placentas when compared with those from the normal control placentas. Together, PLAC8 is a new marker for iEVTs and plays an important role in promoting trophoblast invasion and migration.

PLAC1 is involved in human trophoblast syncytialization.

Placenta specific protein 1 (PLAC1) is thought to be important for murine and human placentation because of its abundant expression in placenta; however, the trophoblast subtypes that express PLAC1 at the fetomaternal interface and the major role of PLAC1 in placentation are still unclear. This study investigated the expression pattern of PLAC1 at the human fetomaternal interface and its involvement in trophoblast syncytialization. Localization of PLAC1 at the fetomaternal interface was studied using in situ hybridization (ISH) and immunohistochemistry (IHC) assays. Real time RT-PCR and Western Blot were employed to exhibit the expression pattern of PLAC1 during human spontaneous syncytialization of term primary cytotrophoblast cells (CTBs). Spontaneous syncytialization of a primary term CTBs model transfected with siRNA specific to PLAC1 was used to investigate the role of PLAC1 during human trophoblast syncytialization. The results showed that PLAC1 was mainly expressed in the human villous syncytiotrophoblast (STB) layer throughout gestation, and the expression level of PLAC1 was significantly elevated during human trophoblast syncytialization. Down-regulation of PLAC1 via specific PLAC1 siRNA transfection attenuated spontaneous syncytialization of primary term CTBs (p<0.05) as indicated by cell fusion index and the expression patterns of the corresponding markers. These data demonstrate the facilitative role of PLAC1 in normal human trophoblast syncytialization.

Role of placenta-specific protein 1 in trophoblast invasion and migration.

Placenta-specific protein 1 (PLAC1), a placenta-specific gene, is known to be involved in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration are investigated by technical studies including real-time RT-PCR, in situ hybridization, immunohistochemistry, and functional studies by utilizing cell invasion and migration assays in the trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblasts (EVTs). The results show that PLAC1 is mainly detected in the trophoblast columns and syncytiotrophoblast of the first-trimester human placental villi, as well as in the EVTs that invade into the maternal decidua. Knockdown of PLAC1 by RNA interference significantly suppresses the invasion and migration of HTR8/SVneo cells and shortens the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the above data suggests that PLAC1 plays an important role in human placental trophoblast invasion and migration.

Transmission of porcine circovirus type 2b (PCV2b) in Kunming mice.

To investigate porcine circovirus type 2b (PCV2b) transmission by contact and vertical infection in Kunming mice (an outbred mouse stock deriving from Swiss albino mice with a high ratio of gene heterozygosis), four mice in cage 6 were inoculated with PCV2b and 25 mice without any treatment were placed into cages 1 to 5 (five mice in each cage). Seven days after being infected, the PCV2-binoculated mice were co-mingled with non-inoculated mice from cages 1 to 5 successively at 7, 14, 21, 28 and 35 days post infection (dpi), respectively, for 3 days. In addition, eleven pregnant mice were injected with PCV2b. Samples were collected from non-inoculated mice and three newborn mice from each litter for PCV2b detection by polymerase chain reaction (PCR) and immunohistochemistry (IHC). The PCR results showed that PCV2b transmission rate among mice in cages 1, 2, 3, 4 and 5 was 0/5, 2/5, 5/5, 5/5 and 1/5, respectively. PCV2b antigen signals generally appeared in most organs of the non-inoculated mice in which viruses were detected by PCR. PCV2b DNA was also detected in newborn mice of PCV2b-infected litters, and viral antigen signals were observed in their organs as well. PCV2b was transmitted in Kunming mice by contact, and it also caused vertical infection through the placenta.

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats.

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n=18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 µg (T1, T2 and T3, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P<0.05) in T3 than in either T1 or T2 (0.341±0.123, 0.236±0.068, and 0.251±0.077, respectively, mean±SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P<0.05) in T3, and were higher (P<0.05) in T1 and T2 than in control groups. For antibody-positive rats, there was a correlation (P<0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T3 group (46.00±4.65) was higher (P<0.05) than in two control groups (29.25±3.72 and 27.92±3.48), and also higher (P<0.05) than in T1 and T2 (37.17±4.99 and 38.75±7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75±4.23 vs. 35.60±3.38, P<0.05). Moreover, litter size and number of placentas were increased (P<0.05) in the pcISI immunization groups, except for the T1 group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 µg yielded the best immune response.

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.

[Cloning and bioinformatics analysis of SLA-DR genes in Hunan Daweizi pigs].

AIM: To evaluate the potential of Daweizi pigs as xenotransplantation dnors from pigs to humans by analyzing the characteristics of SLA-DR genes in Hunan Daweizi pigs. METHODS: SLA-DRA and SLA-DRB genes were amplified by RT-PCR, cloned into pUCm-T vectors, sequenced and analyzed through BLAST in NCBI and related software in ExPASY. RESULTS: The SLA-DRA and SLA-DRB genes were 1 177 bp and 909 bp nucleotides in length, which contain opening reading frame (ORF) and encode 252 and 266 amino acids respectively. Comparing the SLA-DRA and SLA-DRB genes with their counterpart sequences of human, the homologies of amino acid sequences were 82% and 73% respectively. The amino acids in SLA DR alpha chain of Daweizi pigs from position 124 to 136, which bind to human CD4, showed only two differences with HLA DRA: a lle-Val change at position 127 and a Ser-Thr change at position 136. The amino acids in SLA DR beta chain of Daweizi pigs from position 134 to 148, which bind to human CD4, were identical with HLA-DRB. Further comparison with SLA sequences published in GenBank indicated that SLA-DRB gene found in Daweizi pigs has polymorphism while the homology of SLA-DRA gene is up to 100%. CONCLUSION: The cloned SLA-DRA and SLA-DRB in Hunan Daweizi pigs has high polymorphism with HLA-DRA and HLA-DRB in Human, indicates that Daweizi pigs have some advantages as xenotransplantation dnors from pigs to humans.

[Cloning and bioinformatics analysis of SLA-DR genes in Hunan Shaziling pigs].

In order to clone class II DRA and DRB genes of swine leukocyte antigen (SLA) in Hunan Shaziling pigs, to analyze their characteristics and polymorphism and to provide immunological basic parameters for xenotransplantation from pigs to humans. SLA-DRA and SLA-DRB genes in two Shaziling pigs with the absence of porcine endogenous retrovirus (PERV) env-c were amplified by RT-PCR, cloned into PUCm-T vectors, sequenced and analyzed through BLAST in NCBI and related software in ExPASY. The obtained SLA-DRA and SLA-DRB genes of Shaziling pigs were 1,177 and 909 nucleotides in length with their accession numbers in Genbank as EF143987 and EF143988. Bioinformatics analyses have shown that they both contain opening reading frame (ORF) and encode 252 and 266 amino acids respectively. Comparing the ORF and protein sequences of the Shaziling SLA-DRA and SLA-DRB genes with their counterpart sequences of human, the homologies of nucleotide sequences were 83% and 83%, and the homologies of amino acid sequences 83 % and 79% respectively. Further comparison with SLA sequences published in GenBank indicated that SLA-DRB gene found in Shaziling pigs has polymorphism while the homology of SLA-DRA gene is up to 100 % .

[Effects of exogenous metallothionein on thermoresistance and SOD gene expression of dairy cattle].

To approach the effects of exogenous metallothionein (Zn-MT) on the thermoresistance and SOD gene expression of dairy cattle, an experiment was conducted with 28 lactating cows, which were randomly allocated to groups A, B, C and D, and supplemented with 0, 6.0, 12.0 and 16.0 mg Zn-MT x capita(-1), respectively, by intravenous injection. The results showed that the pulse, breath rate, and serum MDA content of the cows in groups B, C and D were lower (P < 0.05 or P < 0.01), while their milk yield, serum- and milk MT contents, blood GSH-PX activity, erythrocyte SOD activity, and SOD gene expression level were higher (P < 0.05 or P < 0.01) than those in group A. All the test indices of the cows in groups C and D were superior (P < 0.05 or P > 0.05) than those in group B, but no significant difference (P > 0.05) was observed between groups C and D. Exogenous Zn-MT had the best effects on the thermoresistance and SOD gene expression of dairy cattle 30 days after injection. All of these suggested that exogenous Zn-MT should be a physiologically active substance effective to the thermoresistance and SOD mRNA expression of dairy cattle, and presented time- and dose-dependent effects.

[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.

[Research of porcine endogenous retrovirus in Shaziling pigs].

To provide basic parameters of evaluating the biological safety of xenotransplantation from pig to human, ear tissues from 31 individuals were randomly collected from a Shazi Ling pig population. PCR and RT-PCR were performed to detect porcine endogenous retrovirus (PERV) proviral DNA and mRNA respectively. The sensitivity of the PCR was evaluated using a positive control. To study tissue distribution, RT-PCR of pol, gag and env was performed in the kidney, heart, liver, lung and spleen of 3 individuals. Finally, env-A, env-B and env-C were amplified, sequenced and analyzed using the BLAST software in NCBI (National Center for Biotechnology Information). The results showed PERV proviral DNA and mRNA could be detected in all 31 individuals by PCR and RT-PCR, respectively. env-A, env-B and env-C were only detected in 2 individuals, while in the other 29 individuals, only env-A and env-B but not env-C was detected. The quantity of DNA in PCR amplification of PERV genes should be more than 15 ng. RT-PCR results showed gag, pol, env-A and env-B were expressed in the kidney, heart, liver, lung and spleen of all 3 individuals, but env-C was not. Sequencing of env genes in Shazi Ling pigs revealed that while there was no difference in env-A sequence when compared to other herd in GenBank, there were 2 and 10 bp differences in the sequences of env-B and env-C respectively, suggesting that env gene is polymorphic in different pig strains. PERV exists in the Shazi Ling pig population and the predominant subtype is PERV-A, B. The distribution of PERV displays no significant tissue specificity and env-C is absent in 93.5% (29/31) of the individuals. The results indicate that Shazi Ling pig may have great potential value as a candidate in xenotransplantation.

[Porcine endogenous retrovirus in Daweizi pigs in Hunan].

OBJECTIVE: To detect porcine endogenous retrovirus (PERV) in Daweizi pigs and to provide basic parameters of evaluating the biological safety for xenotransplantation from pigs to humans. METHODS: Ear tissues from 42 individuals were randomly collected from a Daweizi pig population. PCR and RT-PCR were performed to detect PERV proviral DNA and mRNA respectively. Finally, env-A, env-B, and env-C were amplified, sequenced, and analyzed using the BLAST software in National Center for Biotechnology Information. RESULTS: PERV proviral DNA and mRNA could be detected in the 42 individuals by PCR and RT-PCR, respectively. env-A, env-B and env-C were detected in all the individuals. Compared with other pig species (AY288779, DQ011794 and AY534304), there was 1 and 8 bp differences in the sequences of env-A and env-C, while no difference in env-B. CONCLUSION: PERV exists and has transcriptive activity in Daweizi pigs. The predominate subtype is PERV-ABC. Env genes are firstly cloned and sequenced in Daweizi pigs and there are polymorphism in the breed. As to the biological safety, the breed was not suitable as a donor in xenotransplantation.