摘要：
Peroxisome proliferator-activated receptor (PPAR) gamma has been reported to be implicated in placentation in mice. Previous studies have demonstrated that PPARgamma is also expressed in porcine placenta, primarily localized in vascular endothelial cells (VECs). The present study aimed to investigate the roles of PPARgamma during porcine placental angiogenesis and examine the molecular mechanisms involved in its actions. VECs were incubated with the PPARgamma 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 PPARgamma activation and blockade. The mRNA expression levels of HIF1alpha, HIF2alpha, 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 PPARgamma activation and blockade, whereas VEGF164 mRNA levels remained unaltered. The results of the present study suggested that PPARgamma may mediate porcine placental angiogenesis, by interfering with HIF, VEGF and angiopoietinmediated signaling pathways.
作者机构：
[Juzuo Zhang; Anwen Yuan; Xuan Peng; Zhilong Chen; Xiujun Fan; Jian V Zhang; Qing Yang; Liqun Xue] Laboratory for Reproductive Health,Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences;[Juzuo Zhang; Anwen Yuan; Xuan Peng; Zhilong Chen; Xiujun Fan; Jian V Zhang; Qing Yang; Liqun Xue] Department of Clinic Veterinary Medicine,College of Veterinary Medicine,Hunan Agricultural University
摘要：
Peroxisome proliferator-activated receptor gamma（PPARγ） plays a vital role in placental development of mice and human,but its role and mechanism in pig placentation has not been reported.In the present study,PPARγ expression were investigated in uterus-placenta tissues which were collected from sows on gestation days（GD） 25,40 and 70 using real-time quantitative polymerase chain reaction（qPCR）,Western blot and immunohistochemistry（IHC）.And PPARγ over-expression and deficience were performed in the porcine vascular endothelial cells（VECs） for possible role and mechanism using pIC-neo mammal expression system and CRISPR/Cas9 genome editing system,respectively.Then the angiogenic potential of modified VECs were determined using ICELLegance assay,woud healing assay and capillary-like tube formation assay,followed by determination of the mRNA levels of angiogenic factors,including HIFs-VEGF and angiopoietins signals using qPCR.Results showed that the level of PPARγ mRNA was higher in fetal placenta than that in maternal endometrium on the matched GD40 and 70.In maternal endometrium,expression of PPARγ mRNA was decreased on GD25 and 40 when compared to that of the GD70;in fetal placenta,the level of PPARγ mRNA was also higher on GD40 than that of the GD25,and kept a high level on GD70.The expression of PPARγprotein had a similar trend as its mRNA,kept a higher level on GD40 when compared to those of the GD25 and 70 in both maternal endometrium and fetal placenta.Meanwhile,PPARγ protein was mainly located in the endometrial glandular epithelium and trophoblasts,vascular endothelium and amniotic chorion epithelial cells,with a highest expression in trophoblasts on GD40.In vitro,PPARγ deficience inhibited VECs proliteration and migration,and abrogated the tubes formation.The mRNA levels of HIF1α,HIF2α,VEGF188,Flt1 and Ang-1 were significantly up-regulated in PPARγ over-expression VECs,and Ang-2 with down-regulation.Whereas,the mRNA levels of VEGF isoforms and their receptors were down-regulated in PPARγ null VECs,but HIFs and angiopoietins with insignificantly change.These results suggest that PPARγmediates porcine angiogenesis through VEGF signals,adjustment of VEGF transcription and interaction with their receptors.
摘要：
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. (C) 2016 Published by Elsevier Sp. z o.o. on behalf of Society for Biology of Reproduction& the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn.
通讯机构：
[Xue, Liqun] H;Hunan Agr Univ, Coll Vet Med, Changsha 410128, Hunan, Peoples R China.
关键词：
DBY;antibody;spermatozoa;gender selection
摘要：
DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specific antigen. The DBY gene detected in capacitated mouse sperm codes putative ATP-dependent RNA helicase. The objective was to produce anti-predicted DBY multi-epitope fusion protein antibody, which could be used to determine male specificity of DBY. Epitope prediction is to aid the design of molecules that can mimic structure and function of a genuine epitope, is a useful tool in protein molecule design. This study predicted the DBY epitopes, prepared rabbit poloclonal antibody against DBY multi-epitope fusion protein, then investigated its immunoreactivity. The fusion protein used as the antigen consisted of three regions of DBY with greatest divergence from other family members, cloned together in-frame (with a His tag to facilitate purification). The resulting antibody recognized both the DBY1-2-3 fusion protein and an endogenous DBY protein of the same size. Furthermore, DBY protein was present (Western blot) in testis, male mouse splenocytes and brain, whereas a weaker band was present in the female brain and splenocytes, and finally, ovary produced only a barely visible protein band. Optical density of DBY protein was higher for males versus corresponding tissues from females. Finally, positive signals of DBY1-2-3 antibody were present on only similar to 60% of mature murine sperm (based on immunofluorescent staining and flow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specific epitope present in subpopulations of mouse sperm. Therefore, we concluded that anti-DBY1-2-3 antibody could be an alternative way of producing antibodies to DBY protein. Furthermore, this novel DBY antibody against a multi-epitope artificial antigen has potential for both investigating male-specific binding of DBY and as a new method of sex selection.
