Inhibition of FGF Signalling Pathway Augments the Expression of Pluripotency and Trophoblast Lineage Marker Genes in Porcine Parthenogenetic Blastocyst

The consistent failure to isolate bona fide pluripotent cell lines from livestock indicates that the underlying mechanisms of early lineage specification are poorly defined. Unlike other species, the contrivances of segregation have been comprehensively studied in the mouse. In mouse, FGF/MAPK signalling pathway dictates the segregation of hypoblast (primitive endoderm). However, it is not evident whether this mechanism is also conserved in livestock. Here, in this study, we examined the roles of FGF/MAP kinase signalling pathways in porcine parthenogenetic embryos during the early development. Porcine parthenogenetic embryos were cultured in the medium addition with FGFR inhibitor BGJ398 (10 μm ) or DEMOS. Pluripotency‐ and lineage‐related gene expressions in the early porcine embryos were determined. Compared to control, total cell numbers on day 7 were significantly higher (55 ± 5.96 vs 47 ± 1.97, p < 0.05) in embryos cultured in the presence of BGJ398, but had no significant effect on the rate of blastocyst development (47% vs 44%, p > 0.05). Nonetheless, BGJ398 treatment significantly augmented the expression of pluripotency and trophoblast marker genes (SOX2, OCT4, KLF4 and CDX2), but did not significantly change the expression of NANOG and hypoblast marker gene (GATA4). Furthermore, the addition of FGF signalling agonist (FGF2) during the embryo development significantly decreased the expression of pluripotency and trophoblast marker genes (SOX2, NANOG, KLF4 and CDX2), but no significant effect on the expression of OCT4 and GATA4 was observed. Here, we exhibit that inhibition of FGF signalling could improve the quality of the porcine embryo and escalate the chance to capture pluripotency. Besides, it also promotes the trophoblast development of porcine parthenogenetic embryo. In addition, the data suggested that FGF signalling pathway is dispensable for the segregation of hypoblast and epiblast lineages in porcine embryo during the early development.     

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.     

Significance of CCN2 expression in bovine preimplantation development

In mammalian preimplantation development, the first cell lineage segregation occurs during the blastocyst stage, when the inner cell mass and trophectoderm (TE) differentiate. Species‐specific analyses are essential to elucidate the molecular mechanisms that underlie this process, since they differ between various species. We previously showed that the reciprocal regulation of CCN2 and TEAD4 is required for proper TE differentiation in bovine blastocysts; however, the function of CCN2 during early embryogenesis has remained otherwise elusive. The present study assessed the spatiotemporal expression dynamics of CCN2 in bovine embryos, and evaluated how changes to CCN2 expression (using a CCN2 knockdown (KD) blastocyst model) regulate the expression of pluripotency‐related genes such as OCT4 and NANOG. The conducted quantitative PCR analysis revealed that CCN2 mRNA was expressed in bovine oocytes (at the metaphase stage of their second meiosis) and embryos. Similarly, immunostaining detected both cytoplasmic and nuclear CCN2 at all analyzed oocyte and embryonic stages. Finally, both OCT4 and NANOG expression levels were shown to be significantly reduced in CCN2 KD blastocysts. Together, these results demonstrate that bovine CCN2 exhibits unique expression patterns during preimplantation development, and is required for the proper expression of key regulatory genes in bovine blastocysts.     

Lipid-rich bovine serum albumin improves the viability and hatching ability of porcine blastocysts produced in vitro

The effects of lipid-rich bovine serum albumin (LR-BSA) on the development of porcine blastocysts produced in vitro were examined. Addition of 0.5 to 5 mg/ml LR-BSA to porcine blastocyst medium (PBM) from Day 5 (Day 0 = in vitro fertilization) significantly increased the hatching rates of blastocysts on Day 7 and the total cell numbers in Day-7 blastocysts. When Day-5 blastocysts were cultured with PBM alone, PBM containing LR-BSA, recombinant human serum albumin or fatty acid-free BSA, addition of LR-BSA significantly enhanced hatching rates and the cell number in blastocysts that survived compared with other treatments. The diameter, ATP content and numbers of both inner cell mass and total cells in Day-6 and Day-7 blastocysts cultured with PBM containing LR-BSA were significantly higher than in blastocysts cultured with PBM alone, whereas LR-BSA had no effect on mitochondrial membrane potential. The mRNA levels of enzymes involved in fatty acid metabolism and β-oxidation (ACSL1, ACSL3, CPT1, CPT2 and KAT) in Day-7 blastocysts were significantly upregulated by the addition of LR-BSA. The results indicated that LR-BSA enhanced hatching ability and quality of porcine blastocysts produced in vitro, as determined by ATP content, blastocyst diameter and expression levels of the specific genes, suggesting that the stimulatory effects of LR-BSA arise from lipids bound to albumin.     

Chitosan nanoparticles enhance developmental competence of in vitro-matured porcine oocytes

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus–oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50 µg/ml) during in vitro maturation (IVM). Oocytes treated with 25 µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.     

OCT4 Expression in Outgrowth Colonies Derived from Porcine Inner Cell Masses and Epiblasts

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM‐ and epiblast‐derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)‐like morphology. A total of 104 zona pellucida‐enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC‐like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC‐like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC‐like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT‐PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC‐like morphology. In conclusion, we have established a robust system for derivation of ESC‐like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC‐like morphology although this relationship is lost during early passages.     

Molecular cloning and expression of FGF2 gene in pre‐implantation developmental stages of in vitro‐produced sheep embryos

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO₂, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO₂ incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.     

Effect of short‐term exposure of cumulus–oocyte complex to 3‐morpholinosydnonimine on in vitro embryo development and gene expression in cattle

Short‐term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short‐term exposure of bovine mature cumulus–oocyte complex (COC) to 3‐morpholinosydnonimine (SIN‐1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN‐1 (0, 0.1, 1, 10 and 100 μM SIN‐1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN‐1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short‐term exposure of mature bovine COCs to SIN‐1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.     

Early development of porcine parthenogenetic embryos and reduced expression of primed pluripotent marker genes under the effect of lysophosphatidic acid

To further promote the early development of porcine embryos and capture “naïve” pluripotent state within blastocyst, the experiment explored the effects of lysophosphatidic acid (LPA) on the early development of porcine parthenogenetic embryos and the expression of pluripotency relevant genes. The results showed that the addition of 50 μM LPA significantly improved parthenogenetic embryo cleavage rate (82.7% vs. 74.7%, p < 0.05), blastocyst rate (24.5% vs. 11.3%, p < 0.05) and blastocyst cell count (56 ± 7.9 vs. 42 ± 1.0, p < 0.05) than that of the control group. In addition, immunostaining experiment determined that the fluorescence intensity of OCT4 was also significantly higher than that of the control group. The quantitative real‐time polymerase chain reaction (qRT–PCR) test revealed that addition of 50 μM LPA could significantly enhance the expression level of pluripotent gene OCT4 and trophoblast marker genes CDX2, however, decrease the expression of primitive hypoblast marker gene GATA4. The results also indicated that LPA might decrease the expression of GATA4 through the ROCK signalling pathway. For further investigating the effect of the addition of LPA on the expression of “primed” and “naïve” genes, we also detected the expression of those pluripotency‐related genes by qRT–PCR. The results showed addition of LPA had no significant effect on the expression of “naïve” pluripotent genes, but it was able to significantly decrease the expression of “primed” pluripotent genes, NODAL and Activin‐A; furthermore, it also could significantly improve the expression of OCT4 and c‐Myc which act as two important ES cell renewal factors. Above all, the addition of LPA can facilitate the early development of porcine parthenogenetic embryos, which may be able to benefit for capturing “naïve” pluripotency in vitro through inhibiting “primed” pluripotency.     

Effects of FGF10 on Bovine Oocyte Meiosis Progression,Apoptosis, Embryo Development and Relative Abundance of Developmentally Important Genes In Vitro

Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion‐related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM‐199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real‐time RT‐PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.     

Dihydromyricetin supplementation during in vitro culture improves porcine oocyte developmental competence by regulating oxidative stress

Dihydromyricetin (DHM), a dihydroflavonoid compound, exhibits a variety of biological activities, including antitumor activity. However, the effects of DHM on mammalian reproductive processes, especially during early embryonic development, remain unclear. In this study, we added DHM to porcine zygotic medium to explore the influence and underlying mechanisms of DHM on the developmental competence of parthenogenetically activated porcine embryos. Supplementation with 5 μM DHM during in vitro culture (IVC) significantly improved blastocyst formation rate and increased the total number of cells in porcine embryos. Further, DHM supplementation also improved glutathione levels and mitochondrial membrane potential; reduced natural reactive oxygen species levels in blastomeres and apoptosis rate; upregulated Nanog, Oct4, SOD1, SOD2, Sirt1, and Bcl2 expression; and downregulated Beclin1, ATG12, and Bax expression. Collectively, DHM supplementation regulated oxidative stress during IVC and could act as a potential antioxidant during in vitro porcine oocytes maturation.     

小鼠四倍体囊胚凋亡及凋亡基因的表达

为了更全面的对四倍体胚胎的发育能力进行评估,本研究利用原位末端标记(TUNEL)法检测了电融合生产的小鼠四倍体囊胚细胞凋亡情况,同时用实时定量PCR方法检测了促凋亡基因Bax和抑凋亡基因Bcl-2在四倍体囊胚中的表达。结果显示:四倍体与体外二倍体胚胎发育到囊胚的比率没有显著差别(P0.05);四倍体囊胚无论是内细胞团细胞数、滋养层细胞数还是内细胞团/总细胞数比率都显著低于体内与体外二倍体囊胚(P0.05);四倍体囊胚细胞凋亡指数显著高于体内与体外二倍体囊胚细胞凋亡指数(P0.05);促凋亡基因Bax在四倍体囊胚中的表达显著高于体内与体外二倍体囊胚中的表达(P0.05),而抑凋亡基因Bcl-2在四倍体囊胚中的表达同体内与体外二倍体囊胚中的表达没有显著差异(P0.05)。因此,四倍体囊胚质量比体内与体外二倍体囊胚质量都差,这可能是四倍体胚胎不能发育到足月的原因之一。     

Ascorbic Acid Improves the Developmental Competence of Porcine Oocytes After Parthenogenetic Activation and Somatic Cell Nuclear Transplantation

In this study, a dose-response assessment was performed to understand the relation between supplementation of media with L-ascorbic acid or vitamin C and porcine oocyte maturation and the in vitro development of parthenotes (PA) and handmade cloned (HMC) embryos. Various concentrations (0, 25, 50 and 100 µg/ml) of vitamin C supplemented in in vitro maturation (IVM) and culture (IVC) media were tested. None of these vitamin C additions affected nuclear maturation of oocytes, yet supplementation at 50 µg/ml led to significantly increased intracellular glutathione (GSH) levels and reduced reactive oxygen species (ROS). When cultured in IVM- and/or IVC-supplemented media, the group supplemented with 50 µg/ml of vitamin C showed improved cleavage rates, blastocyst rates and total cell numbers per blastocyst (P<0.05) compared with other groups (control, 25 µg/ml and 100 µg/ml). In contrast, supplementation with 50 µg/ml vitamin C decreased (P<0.05) the apoptosis index as compared with the groups supplemented with 100 µg/ml. In addition, even with a lower blastocyst rate to start with (37.6 vs. 50.3%, P<0.05), supplementation of HMC embryos with vitamin C ameliorated their blastocyst quality to the extent of PA embryos as indicated by their total cell numbers (61.2 vs. 59.1). Taken together, an optimized concentration of vitamin C supplementation in the medium not only improves blastocyst rates and total cell numbers but also reduces apoptotic indices, whereas overdosages compromise various aspects of the development of parthenotes and cloned porcine embryos.