Effect of bromodomain PHD-finger transcription factor (BPTF) on trophoblast epithelial-to-mesenchymal transition.

The trophoblast epithelial-to-mesenchymal transition (EMT) is a procedure related to embryo implantation, spiral artery establishment and fetal-maternal communication, which is a key event for successful pregnancy. Inadequate EMT is one of the pathological mechanisms of recurrent miscarriage (RM). Whole-exome sequencing revealed that the mutation of bromodomain PHD-finger transcription factor (BPTF) was strongly associated with RM. In the present study, the effects of BPTF on EMT and the underlying mechanism were investigated. We found that the expression of BPTF in the villi of RM patients was significantly downregulated. Gene Ontology (GO) analysis revealed that BPTF participated in cell adhesion. The knockdown of BPTF prevented EMT and attenuated trophoblast invasion in vitro. BPTF activated Slug transcription by binding directly to the promoter region of the Slug gene. Interestingly, the protein levels of both Slug and BPTF were decreased in the villous cytotrophoblasts (VCTs) of RM villi. In conclusion, BPTF participates in the regulation of trophoblast EMT by activating Slug expression, suggesting that BPTF defects are an important factor in RM pathogenesis.

The Prl3d1-Cre mouse line selectively induces the expression of Cre recombinase in parietal trophoblast giant cells.

The placenta plays a pivotal role in the maintenance of normal pregnancy, but how it forms, matures, and performs its function remains poorly understood. Here, we describe a novel mouse line (Prl3d1-iCre) that expresses iCre recombinase under the control of the endogenous prl3d1 promoter. Prl3d1 has been proposed as a marker for distinguishing trophoblast giant cells (TGCs) from other trophoblast cells in the placenta. The in vivo efficiency and specificity of the Cre line were analyzed by interbreeding Prl3d1-iCre mice with B6-G/R reporter mice. Through anatomical studies of the placenta and other tissues of Prl3d1-iCre/+; B6-G/R mouse mice, we found that the tdTomato signal was expressed in parietal trophoblast giant cells (P-TGCs). Thus, we report a mouse line with ectopic Cre expression in P-TGCs, which provides a valuable tool for studying human pathological pregnancies caused by implantation failure or abnormal trophoblast secretion due to aberrant gene regulation.

The chromatin remodeling protein BPTF mediates cell cycle, proliferation and apoptosis in porcine ovarian granulosa cells.

Bromodomain PHD finger transcription factor (BPTF), a core subunit of nucleosome-remodeling factor (NURF) complex, plays an important role in chromatin remodeling. However, few information of BPTF is available in pig, especially in mammalian follicular granulosa cells (GCs). The present study firstly confirmed that BPTF in porcine was relative close to human and mouse. The expression of BPTF could be detected in ovary, testes, lung, kidney, large intestine, and small intestine. And a relative high expression of BPTF was observed in ovarian follicles and GCs. When BPTF was knocked down (BPTF-siRNA), the viability of GCs was affected. And the expression level of CDK1, cyclin B1, CDK4 and CDK2 was higher than the control, which might indicate that the cell cycle of GCs was inhibited from S to G2/M phase. Although the apoptosis level was induced in the BPTF-siRNA GCs, the reduced level of H3K4 methylation was detected with the down regulation of SMYD3, EHMT2 and DPY30. Thereby, results in the present might provide the primary knowledge of BPTF in GCs and the follicular development in pig.

Proteomics reveals the underlying mechanism by which the first uneven division affects embryonic development in pig.

One of the most typical abnormal cleavage patterns during early embryonic development is uneven division, but the first uneven division of pig zygote is common. Uneven division results in different daughter cell sizes and an uneven distribution of organelles such as lipid droplet, mitochondria, but the developmental capacity of daughter cells and proteomic changes of daughter cells are still unclear. Therefore, the developmental ability and proteomic quantification were investigated on blastomeres from even division (ED) or uneven division (UD) embryos at 2-cell stage in the present study. Firstly, the developmental ability was affected by the blastomeric size, when compared with medium blastomeres (MBs), the large blastomeres (LBs) with the higher cleavage rate but the small blastomeres (SBs) with the lower rate was observed. Subsequently, proteomic analysis was performed on blastomeres of LBs, MBs and SBs, a total of 109 DEPs were detected, which were involved in protein metabolism and processing, energy metabolism and ribosome. In particular, DEPs in LBs vs. SBs were focused on RNA binding and actin cytoskeletal tissue. Two protein-dense networks associated with RNA binding and cytoskeleton were revealed by further protein-protein interaction (PPI) analysis of DEPs in LBs vs. SBs, that DDX1 related to RNA binding and ACTB related to cytoskeleton were confirmed in UD embryos. Therefore, a briefly information of DEPs in blastomeres of 2-cell stage pig embryos was described in the present study, and it further confirmed that the formation of uneven division of the first cell cycle of pig embryos might be controlled by the cytoskeleton; the developmental capacity of daughter cells might be affected by the energy metabolism, RNA binding and ribosome, and further account for the developmental potential of the whole embryo.

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging.

Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased ß-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

Astaxanthin alleviates palmitic acid-induced hindrance of porcine oocyte maturation.

Increased palmitic acid (PA) levels have been found in females with reduced fertility due to metabolic disorders. However, effective antioxidant astaxanthin (AXE) might positively affect animal reproduction. Therefore, the present study was designed to evaluate the impact of a high concentration of PA on oocyte maturation and the possible protective effect of AXE against high PA concentration in pigs. Firstly, different concentrations (0.2, 0.5, 0.8 mM) of PA were conducted on in vitro maturation (IVM) of pig oocytes (PA0.2, PA0.5, and PA0.8), while no addition of PA was performed as the control group (Ctrl). Results showed that the cumulus cell expansion index (CCEI) was lower in PA0.5 and PA0.8 groups compared to Ctrl group (p < .05). In PA0.5 group, not only did the percentage of matured oocytes with the first polar body (PB1) reduced, that with more oocytes arrested at germinal vesicle (GV) stage (53.44% ± 7.16% vs. 20.93% ± 5.16%, p < .05), but also a higher number of transzonal projections (TZPs) was observed in PA0.5 than Ctrl group. Besides, supplement of PA resulted in a dose-dependent decrease in mitochondrial activity. Although no difference of lipid content was observed between PA0.5 and Ctrl groups, the lipid content was at a higher level in PA0.2 group than in the other three groups. Hence, concentration of 0.5 mM of PA was performed in the following experiments, and 2.5 µM AXE carried out to investigate the possible relief effects of PA (PA0.5 + AXE). Results showed that the percentage of matured oocytes with PB1 was higher in PA0.5 + AXE than in PA0.5 group (63.43% ± 1.50% vs. 55.33% ± 0.81%, p < .01), and ROS levels both in oocytes and their cumulus cells (CCs) reduced in PA0.5 + AXE when compared to PA0.5 group. In addition, the rate of CCs with apoptosis decreased in PA0.5 + AXE, and the expression level of caspase 3 and BAX was lower than PA0.5 group. In conclusion, the maturation of pig oocytes was inhibited by the high concentration of PA; however, this negative effect of PA-induced might be relieved by the supplement of AXE.

Optimal Stage for Cryotop Vitrification of Porcine Embryos.

Different development stages of porcine embryos have different tolerance to low temperature. Therefore, we took the porcine embryos after parthenogenetic activation (PA) as the model, to explore the optimal development stage for vitrification during morula (D4), early blastocyst (D5), and expanded blastocyst (D6) after PA (D0). Embryos were observed with microscope and analyzed by different staining after cryo-recovery for 24 hours. The quality of embryos was damaged after vitrification, including embryonic nuclei, DNA, cytoskeleton, and organelles. The re-expansion rate at 24 hours of D5 embryos was significantly higher than those of D4 and D6 embryos (D5 vs. D4 vs. D6, 27.620 ± 0.041 vs. 7.809 ± 0.027 vs. 13.970 ± 0.032, p < 0.05). Therefore, D5 embryos were selected as research objects to explore the effect of vitrification on lipid in vitrified embryos. The results showed that the expression levels of perilipin PLIN3 messenger RNA (mRNA) and triacylglycerol synthesis-related genes AGPAT1 and DGAT mRNA are significantly reduced (p < 0.05). Vitrification affected lipid synthesis, which might have an irreversible impact on embryonic development. In conclusion, our data demonstrated that the optimal stage of vitrification was D5 for early blastocysts.

Division behaviours and their effects on pre-implantation development of pig embryos.

The quality of pre-implantation embryos could affect developmental efficiency after embryo transfer. However, the assessment of pre-implantation embryos was unsatisfactory, especially in pig embryos to date. Therefore, this study was designed to investigate available and applicable parameters that indicate developmental potential and quality of porcine pre-implantation embryos produced by handmade cloning (HMC), and parthenogenetic activation without zona pellucida (PAZF) and with zona pellucida (PAZI). Firstly, a common division behaviour was detected, that is the formation of uneven division with two unequal size blastomeres (UD 2-cell), especially in HMC embryos; then, the proportion of UD 2-cell was found to be significantly higher than that of even division with equal size blastomeres (ED 2-cell) (72.56 ± 4.56 vs. 24.57 ± 1.92). The formation of UD 2-cell might be due to the spindle migration along the long axis in 1-cell stage, and the cleavage furrow was not formed in the centre of cytoplasm. In the two sister blastomeres of UD 2-cell, uneven distribution of organelles (mitochondria and lipid droplet) was observed with lower proportion in the smaller one (p < .05). Although no difference in blastocyst rate was observed between UD and ED 2-cell embryos, the cell number per blastocyst from UD 2-cell embryos was lower than that from ED 2-cell embryos (44.15 ± 2.05 vs. 51.55 ± 1.83). Besides, because of non-synchronized division of each blastomere, the following three cleavage routes were observed in all HMC/PAZF/PAZI embryos: T1 (2-cell → 3-cell → 4-cell → ≥5-cell → morula → blastocyst), T2 (2-cell → 3-cell → 4-cell → morula → blastocyst) and T3 (2-cell → 3-cell/4-cell → morula → blastocyst). Therefore, in pig in vitro-produced embryos, division behaviours of uneven volume of cytoplasm and non-synchronized cell cycles were observed at the early embryonic developmental stage, which might be another potential factor to evaluate embryonic development.

Dimensional properties of Laguerre-Gaussian vortex beams.

We derive and analyze the dimensional properties of Laguerre-Gaussian vortex beams theoretically and numerically. The analytical expressions of inner and outer radii are given out and proved to be proportional to the transverse beam size, when the topological charge remains. The ratio of the outer and inner radius only depends on the topological charge, having nothing to do with the waist radius and the propagation distance, and the ratio decreases as the topological charge increases. Using a spatial light modulator loaded with computer-generated holograms, we generate vortex beams. The experimental results are in good agreement with our numerical simulations. This research may provide useful insights into the study of the optical vortex beam and its further applications.