The patient-specific mouse model with Foxg1 frameshift mutation uncovers the pathophysiology of FOXG1 syndrome.

Single allelic mutations in the gene encoding the forebrain-specific transcription factor FOXG1 lead to FOXG1 syndrome (FS). Patient-specific animal models are needed to understand the etiology of FS, as FS patients show a wide spectrum of symptoms correlated with location and mutation type in the FOXG1 gene. Here we report the first patient-specific FS mouse model, Q84Pfs heterozygous (Q84Pfs-Het) mice, mimicking one of the most predominant single nucleotide variants in FS. Intriguingly, we found that Q84Pfs-Het mice faithfully recapitulate human FS phenotypes at the cellular, brain structural, and behavioral levels. Importantly, Q84Pfs-Het mice exhibited myelination deficits like FS patients. Further, our transcriptome analysis of Q84Pfs-Het cortex revealed a new role for FOXG1 in synapse and oligodendrocyte development. The dysregulated genes in Q84Pfs-Het brains also predicted motor dysfunction and autism-like phenotypes. Correspondingly, Q84Pfs-Het mice showed movement deficits, repetitive behaviors, increased anxiety, and prolonged behavior arrest. Together, our study revealed the crucial postnatal role of FOXG1 in neuronal maturation and myelination and elucidated the essential pathophysiology mechanisms of FS.

Physiological and Functional Roles of Neurotrophin-4 During In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15, sperm-oocyte interaction regulator CD9, and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.

Exploring the mechanism of trehalose: dual functions of PI3K/Akt and VPS34/mTOR pathways in porcine oocytes and cumulus cells†.

Autophagy, an intracellular recycling system, is essential for the meiotic maturation of porcine oocytes. Trehalose has been reported as a novel mammalian target of rapamycin (mTOR)-independent autophagy inducer in many cells. Furthermore, we previously have demonstrated that trehalose supplementation during in vitro maturation of porcine oocytes improves the developmental competence of parthenogenetic embryos, possibly via autophagic activation, whereas the underlying mechanisms remain unclear. Therefore, the aim of this study was to address this issue. We found that trehalose plays a role as an autophagy activator by autophagic flux assay and determined that it promotes phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) inhibition and vacuolar protein sorting 34 (VPS34)/mTOR activation by immunoblotting, both in cumulus cells (CCs) and oocytes. However, interestingly, the effects and the mechanisms regulated by trehalose were different in them, respectively. In CCs, the autophagy was activated through the improvement of lysosomal function/autophagic clearance viability by upregulation of coordinated lysosomal expression and regulation genes via PI3K/Akt inhibition. Whereas in oocytes, autophagy was activated via induction of VPS34, which directly influences autophagosome formation, and the precise meiotic process was ensured via Akt inhibition and mTOR activation. Taken together, this study furtherly elucidates the novel detailed mechanism of trehalose during porcine oocyte maturation, thus laying the biological foundations for pharmacological application.

Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRASG12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.

Beneficial Effects of Neurotrophin-4 Supplementation During in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Embryonic Development After Parthenogenetic Activation.

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75NTR) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.

Establishment of 3D Neuro-Organoids Derived from Pig Embryonic Stem-Like Cells.

Although the human brain would be an ideal model for studying human neuropathology, it is difficult to perform in vitro culture of human brain cells from genetically engineered healthy or diseased brain tissue. Therefore, a suitable model for studying the molecular mechanisms responsible for neurological diseases that can appropriately mimic the human brain is needed. Somatic cell nuclear transfer (SCNT) was performed using an established porcine Yucatan EGFP cell line and whole seeding was performed using SCNT blastocysts. Two Yucatan EGFP porcine embryonic stem-like cell (pESLC) lines were established. These pESLC lines were then used to establish an in vitro neuro-organoids. Aggregates were cultured in vitro until 61 or 102 days after neural induction, neural patterning, and neural expansion. The neuro-organoids were sampled at each step and the expression of the dopaminergic neuronal marker (TH) and mature neuronal marker (MAP2) was confirmed by reverse transcription-PCR. Expression of the neural stem cell marker (PAX6), neural precursor markers (S100 and SOX2), and early neural markers (MAP2 and Nestin) were confirmed by immunofluorescence staining. In conclusion, we successfully established neuro-organoids derived from pESLCs in vitro. This protocol can be used as a tool to develop in vitro models for drug development, patient-specific chemotherapy, and human central nervous system disease studies.

Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation.

Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs.

This study aimed to examine the effects of treatment with glucuronic acid (GA) and N-acetyl-D-glucosamine (AG), which are components of hyaluronic acid (HA), during porcine oocyte in vitro maturation (IVM). We measured the diameter of the oocyte, the thickness of the perivitelline space (PVS), the reactive oxygen species (ROS) level, and the expression of cumulus cell expansion and ROS-related genes and examined the cortical granule (CG) reaction of oocytes. The addition of 0.05 mM GA and 0.05 mM AG during the first 22 h of oocyte IVM significantly increased oocyte diameter and PVS size compared with the control (non-treatment). The addition of GA and AG reduced the intra-oocyte ROS content and improved the CG of the oocyte. GA and AG treatment increased the expression of CD44 and CX43 in cumulus cells and PRDX1 and TXN2 in oocytes. In both the chemically defined and the complex medium (Medium-199 + porcine follicular fluid), oocytes derived from the GA and AG treatments presented significantly higher blastocyst rates than the control after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). In conclusion, the addition of GA and AG during IVM in pig oocytes has beneficial effects on oocyte IVM and early embryonic development after PA and SCNT.

R-Spondin 2 and WNT/CTNNB1 Signaling Pathways Are Required for Porcine Follicle Development and In Vitro Maturation.

The secretion of oocyte-derived paracrine factors, such as R-spondin2, is an essential mechanism for follicle growth by promoting the proliferation and differentiation of cumulus cells around oocytes. In the present study, we aimed to identify the effect of R-spondin2 during follicular development. First, R-spondin2-related factors (R-spondin2, CTNNB1, LGR4, and LGR5) were identified through immunofluorescence in porcine ovarian tissue. CTNNB1 was expressed in ooplasm, and CTNNB1 and LGR4 were expressed in granulosa cells. In addition, R-spondin2, LGR4, and LGR5 were expressed in the theca interna. These results imply that these proteins play a major role in porcine follicular development. In addition, the effects of R-spondin2 on the in vitro maturation process of porcine cumulus oocyte complexes and subsequent embryonic development were confirmed. A treatment of 100 ng/mL R-spondin2 in the in vitro maturation (IVM) process increased nuclear maturation and increased the expression of EGFR mRNA in cumulus cells. The EGFR-ERK signal is essential for oocyte maturation, ovulation, and luteinization. R-spondin2 treatment also increased the expression of CTNNB1 and EGFR in primary cultured cumulus cells. In conclusion, RSPO2 and WNT/CTNNB1 signaling pathways are required for porcine follicle development and are predicted to be involved in the EGFR-ERK signaling pathway.

The effect of copper supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation.

Copper (Cu) ions have redox activity and act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. In this study, we aimed to examine the effects of copper (II) chloride dihydrate (CuCl2·2H2O) on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA). Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, the mRNA expression levels of various genes, and developmental competence were analyzed. During IVM, the maturation medium was supplemented with various concentrations of Cu (0, 0.7, 1.4, and 2.8 µg/mL). After 42 h of IVM, Cu supplementation significantly increased the number of oocytes in the metaphase II stage. Further, the 1.4 µg/mL Cu group showed significantly higher intracellular GSH levels than the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. The cumulus cell expansion index was higher in the 0.7 and 1.4 µg/mL Cu groups than in the other groups. In the 0.7 µg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher than those in the control group. Moreover, increased levels of Sod1 transcript, correlated with the antioxidative response, were observed in the 0.7 and 1.4 µg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. Upon evaluation of subsequent embryonic development after PA, the 0.7 µg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves porcine oocyte maturation and the subsequent embryonic potential of PA embryos by reducing oxidative stress and apoptosis.

Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs.

Canine induced pluripotent stem cells (ciPSCs) can provide great potential for regenerative veterinary medicine. Several reports have described the generation of canine somatic cell-derived iPSCs; however, none have described the canine somatic cell reprogramming using a non-integrating and self-replicating RNA transfection method. The purpose of this study was to investigate the optimal strategy using this approach and characterize the transition stage of ciPSCs. In this study, fibroblasts obtained from a 13-year-old dog were reprogrammed using a non-integrating Venezuelan equine encephalitis (VEE) RNA virus replicon, which has four reprogramming factors (collectively referred to as T7-VEE-OKS-iG and comprised of hOct4, hKlf4, hSox2, and hGlis1) and co-transfected with the T7-VEE-OKS-iG RNA and B18R mRNA for 4 h. One day after the final transfection, the cells were selected with puromycin (0.5 µg/mL) until day 10. After about 25 days, putative ciPSC colonies were identified showing TRA-1-60 expression and alkaline phosphatase activity. To determine the optimal culture conditions, the basic fibroblast growth factor in the culture medium was replaced with a modified medium supplemented with murine leukemia inhibitory factor (mLIF) and two kinase inhibitors (2i), PD0325901(MEK1/2 inhibitor) and CHIR99021 (GSK3ß inhibitor). The derived colonies showed resemblance to naïve iPSCs in their morphology (dome-shaped) and are dependent on mLIF and 2i condition to maintain an undifferentiated phenotype. The expression of endogenous pluripotency markers such as Oct4, Nanog, and Rex1 transcripts were confirmed, suggesting that induced ciPSCs were in the late intermediate stage of reprogramming. In conclusion, the non-integrating and self-replicating VEE RNA replicon system can potentially make a great contribution to the generation of clinically applicable ciPSCs, and the findings of this study suggest a new method to utilize the VEE RNA approach for canine somatic cell reprogramming.

Transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Growth differentiation factor 8 regulates SMAD2/3 signaling and improves oocyte quality during porcine oocyte maturation in vitro†.

Growth differentiation factor 8 (GDF8), also known as myostatin, is a member of the transforming growth factor-ß (TGF-ß) family and has been identified as a strong physiological regulator of muscle differentiation. Recently, the functional role of GDF8 in reproductive organs has received increased interest following its detection in the human placenta and uterus. To investigate the effects of GDF8 during porcine oocyte in vitro maturation (IVM), we assessed the quality of matured oocytes. Furthermore, we investigated the specific gene transcription and protein activation levels in oocytes and cumulus cells after IVM and subsequent embryonic development after in vitro fertilization and parthenogenetic activation. Prior to these experiments, the concentration of GDF8 in porcine follicular fluid was determined. During the entire IVM period, 1.3 ng/mL GDF8 and its signaling inhibitor SB431542 (SB) at 5 µM were added as control, SB, SB + GDF8, and GDF8 groups, respectively. Our results demonstrate that supplementation with GDF8 during porcine oocyte IVM enhanced both meiotic and cytoplasmic maturation, with altered transcriptional patterns, via activation of Sma- and Mad-related protein 2/3 (SMAD2/3). Using the pharmacological inhibitor SB431542, we demonstrated that inhibition of GDF8-induced Smad2/3 signaling reduces matured oocyte quality. In conclusion, for the first time, we demonstrated paracrine factor GDF8 in porcine follicular fluid in vivo. Furthermore, we showed that GDF8 supplementation improved mature oocyte quality by regulating p38 mitogen-activated protein kinase phosphorylation and intracellular glutathione and reactive oxygen species levels during porcine IVM.

GDF8 enhances SOX2 expression and blastocyst total cell number in porcine IVF embryo development.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-ß family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.

Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium.

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine-induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma- and Mad-related protein (SMAD) inhibitors, a TGF-ß inhibitor and BMP4 inhibitor. The dSMADi-derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid-treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma-cultured medium including injury-related cytokines treated porcine iPSC-NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

Isolation and characterization of GFAP-positive porcine neural stem/progenitor cells derived from a GFAP-CreERT2 transgenic piglet.

BACKGROUND: The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model. RESULTS: Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreERT2 under the control of the GFAP promoter (pGFAP-CreERT2). The isolated pGFAP-CreERT2 NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons. CONCLUSIONS: Collectively, these results indicate that GFAP promoter-driven porcine CreERT2 NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ᅟ.

Embryotropic effects of vascular endothelial growth factor on porcine embryos produced by in vitro fertilization.

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ±â€¯3.94%) and blastomeres (67.18 ±â€¯3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ±â€¯3.94) and blastomeres (23.23 ±â€¯4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.

Lysophosphatidic acid increases in vitro maturation efficiency via uPA-uPAR signaling pathway in cumulus cells.

Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 µM). After 42 h of IVM, the 30 µM LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 µM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 µM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 µM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30 µM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30 µM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 µM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.

Production of transgenic pigs using a pGFAP-CreERT2/EGFP LoxP inducible system for central nervous system disease models.

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT.

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo.

BACKGROUND: Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development. RESULTS: SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses. CONCLUSIONS: Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.