Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 µM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 µM niacin had an increased cleavage rate (p < .05). In addition, 300 µM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 µM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 µM increased glutathione levels in day two embryos. On day seven, 300 µM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 µM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

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.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs.

This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

Soybean-VCF2Genomes: a database to identify the closest accession in soybean germplasm collection.

BACKGROUND: The development of next generation sequencer (NGS) and the analytical methods allowed the researchers to profile their samples more precisely and easier than before. Especially for agriculture, the certification of the genomic background of their plant materials would be important for the reliability of seed market and stable yield as well as for quarantine procedure. However, the analysis of NGS data is still difficult for non-computational researchers or breeders to verify their samples because majority of current softwares for NGS analysis require users to access unfamiliar Linux environment. MAIN BODY: Here, we developed a web-application, "Soybean-VCF2Genomes", http://pgl.gnu.ac.kr/soy_vcf2genome/ to map single sample variant call format (VCF) file against known soybean germplasm collection for identification of the closest soybean accession. Based on principal component analysis (PCA), we simplified genotype matrix for lowering computational burden while maintaining accurate clustering. With our web-application, users can simply upload single sample VCF file created by more than 10x resequencing strategy to find the closest samples along with linkage dendrogram of the reference genotype matrix. CONCLUSION: The information of the closest soybean cultivar will allow breeders to estimate relative germplasmic position of their query sample to determine soybean breeding strategies. Moreover, our VCF2Genomes scheme can be extended to other plant species where the whole genome sequences of core collection are publicly available.

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts.

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC-derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC-derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF-derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell-oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC-derived SCNT embryos showed higher blastocyst formation (48.4%) than FF-derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.

Combined Treatment with Demecolcine and 6-Dimethylaminopurine during Postactivation Improves Developmental Competence of Somatic Cell Nuclear Transfer Embryos in Pigs.

This study determined the effects of postactivation treatment with demecolcine and/or 6-dimethylaminopurine (6-DMAP) on in vivo and in vitro developmental competence of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were treated for 4 hours with 0.4 µg/mL demecolcine, 2 mM 6-DMAP, or both after electric activation, then transferred to surrogate pigs or cultured for 7 days. The formation rate of SCNT embryos with a single pronucleus was higher in combined treatment with demecolcine and 6-DMAP (95.2%) than treatment with demecolcine alone (87.1%). Blastocyst formation of SCNT embryos was significantly increased in combined treatment with demecolcine and 6-DMAP (48.7%) compared with demecolcine (22.2%) or 6-DMAP alone (37.3%). Fluctuation of maturation promoting factor activity showed different patterns among various postactivation treatments. Pregnancy was established in 1 of 5 surrogates after transfer of SCNT embryos that were treated with demecolcine and 6-DMAP. The pregnant surrogate delivered one healthy live piglet. The results of our study demonstrated that postactivation treatment with demecolcine and 6-DMAP together improved preimplantation development and supported normal in vivo development of SCNT pig embryos, probably influencing MPF activity and nuclear remodeling, including induction of single pronucleus formation after electric activation.

In vitro oocyte maturation in a medium containing reduced sodium chloride improves the developmental competence of pig oocytes after parthenogenesis and somatic cell nuclear transfer.

The present study investigated the effects of IVM in hypotonic medium containing reduced (61.6mM) NaCl compared with isotonic medium containing 108.0mM NaCl (designated L and N respectively) on oocyte maturation and embryonic development in pigs. IVM culture was divided into four periods at 11-h intervals. Oocytes cultured in N for 33h and then in L for 11h of IVM (N-N-N-L) showed significantly improved (P<0.05) nuclear maturation of oocytes (75.4-79.0% vs 60.2-85.8%) and blastocyst formation (61.5-66.1% vs 45.2-67.5%) after parthenogenesis (PA) compared with other treatments (L-L-L-L, L-L-L-N, L-L-N-L, N-N-L-L, N-N-L-N, L-L-N-L, L-N-N-L and N-L-N-L). Oocytes matured in L-L-L-L and N-N-N-L had an increased (P<0.05) perivitelline space (11.0-12.5 vs 5.5µm) and intraoocyte reduced glutathione (GSH) content (1.39-1.41 vs 1.00 pixels per oocyte) relative to oocytes matured in N-N-N-N. Somatic cell nuclear transfer (SCNT) embryos derived from the N-N-N-L treatment had significantly (P<0.05) higher blastocyst formation (53.5%) than embryos derived from Medium-199 (37.4%) and N-N-N-N (41.8%) treatments. Overall, the results demonstrate that maturation of pig oocytes in hypotonic medium with reduced NaCl during the last 11h of IVM increases the developmental competence of oocytes after PA and SCNT by improving the cytoplasmic microenvironment, including an increased GSH content in IVM oocytes.

Colcemid treatment during oocyte maturation improves preimplantation development of cloned pig embryos by influencing meiotic progression and cytoplasmic maturation.

The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98.4%) than in controls not exposed to colcemid (68.7%). The proportion of metaphase-II (MII) oocytes after 30 hr of IVM was higher in control (79.6%) than in COL0-22 oocytes (61.7%); overall nuclear progression to the MII stage was not influenced by colcemid treatment by the end of the IVM period (93.8, 86.7, 86.8, and 84.8% for control, COL0-22, COL38-42, and COL0-22/38-42, respectively). COL0-22 oocytes showed higher intra-oocyte glutathione content (1.7 vs. 1.0-1.3 pixels/oocyte) and increased blastocyst formation after PA (68.7% vs. 42.5-52.2%) and SCNT (39.4% vs. 16.3-28.6%) than control, COL38-42, and COL0-22/38-42 oocytes. Colcemid treatment for 0-22 and 0-22/38-42 hr of IVM also stimulated the expression of cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNAs. Our results thus demonstrate that the presence of colcemid during the early stage of IVM stimulates preimplantation development of PA and SCNT porcine embryos by improving the cytoplasmic microenvironment.

Effects of MnTBAP on Porcine Semen Cryopreservation and Capacitation.

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 µM of MnTBAP based on a Tris-egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 µM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 µM and 150 µM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 µM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 µM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 µM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze-thawing, improving the fertilization capacity, and leading to increased embryo development.

Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step.

Introduction: Embryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals. Methods: Oocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze-thawing. Results: The survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05). Conclusion: Our study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze-thawing.

Effects of pupil size in video head-impulse tests.

The results of video head impulse tests (video-HITs) may be confounded by data artifacts of various origins, including pupil size and eyelid obstruction of the pupil. This study aimed to determine the effect of these factors on the results of video-HITs. We simulated ptosis by adopting pharmacological dilatation of the pupil in 21 healthy participants (11 women; age 24-58 years). Each participant underwent video-HITs before and after pupillary dilatation using 0.5% tropicamide. We assessed the changes in the vestibulo-ocular reflex (VOR) gain, corrective saccade amplitude, and frequency of eyelid flicks. After pupillary dilatation, the VOR gain decreased for both right (RAC; 1.12 [Formula: see text] 0.12 vs. 1.01 [Formula: see text] 0.16, p = 0.011) and left anterior canals (LACs; 1.15 [Formula: see text] 0.13 vs. 0.96 [Formula: see text] 0.14, p < 0.001), and right posterior canal (RPC, 1.10 [Formula: see text] 0.13 vs. 0.98 [Formula: see text] 0.09, p = 0.001). The corrective saccade amplitudes also decreased significantly for all four vertical canals. The frequency of eyelid flicks, however, did not change. The changes of VOR gain were positively correlated with the lid excursion in RPC (r = 0.629, p = 0.002) and LPC (r = 0.549, p = 0.010). Our study indicates that eyelid position and pupil size should be considered when interpreting the results of video-HITs, especially for the vertical canals. Pupils should be shrunk in a very well-lit room, and artifacts should be prevented by taping or lifting the eyelids as required during video-HITs.

Establishment of porcine embryonic stem cells in simplified serum free media and feeder free expansion.

BACKGROUND: The establishment of stable porcine embryonic stem cells (pESCs) can contribute to basic and biomedical research, including comparative developmental biology, as well as assessing the safety of stem cell-based therapies. Despite these advantages, most pESCs obtained from in vitro blastocysts require complex media and feeder layers, making routine use, genetic modification, and differentiation into specific cell types difficult. We aimed to establish pESCs with a single cell-passage ability, high proliferative potency, and stable in long-term culture from in vitro-derived blastocysts using a simplified serum-free medium. METHODS: We evaluated the establishment efficiency of pESCs from in vitro blastocysts using various basal media (DMEM/F10 (1:1), DMEM/F12, and a-MEM) and factors (FGF2, IWR-1, CHIR99021, and WH-4-023). The pluripotency and self-renewal capacity of the established pESCs were analyzed under feeder or feeder-free conditions. Ultimately, we developed a simplified culture medium (FIW) composed of FGF2, IWR-1, and WH-4-023 under serum-free conditions. RESULTS: The pESC-FIW lines were capable of single-cell passaging with short cell doubling times and expressed the pluripotency markers POU5F1, SOX2, and NANOG, as well as cell surface markers SSEA1, SSEA4, and TRA-1-60. pESC-FIW showed a stable proliferation rate and normal karyotype, even after 50 passages. Transcriptome analysis revealed that pESC-FIW were similar to reported pESC maintained in complex media and showed gastrulating epiblast cell characteristics. pESC-FIW were maintained for multiple passages under feeder-free conditions on fibronectin-coated plates using mTeSR™, a commercial medium used for feeder-free culture, exhibiting characteristics similar to those observed under feeder conditions. CONCLUSIONS: These results indicated that inhibition of WNT and SRC was sufficient to establish pESCs capable of single-cell passaging and feeder-free expansion under serum-free conditions. The easy maintenance of pESCs facilitates their application in gene editing technology for agriculture and biomedicine, as well as lineage commitment studies.

Pig oocytes with a large perivitelline space matured in vitro show greater developmental competence after parthenogenesis and somatic cell nuclear transfer.

The objective of this study was to examine the developmental competence of pig oocytes in relation to the size of the perivitelline space (PVS) of oocytes matured in vitro. Immature oocytes were matured in medium 199 or porcine zygote medium (PZM)-3 containing 108 or 61.6 mM NaCl. In vitro-matured (IVM) oocytes were examined for intracellular glutathione (GSH) level; cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNA levels; and developmental competence after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). IVM oocytes with a larger PVS had higher (P < 0.05) levels of intracellular GSH (1.00 pixels/oocyte vs. 0.57 pixels/oocyte) and blastocyst formation (54.3% vs. 37.3%) after PA than oocytes with a smaller PVS. Culturing oocytes for maturation in PZM-3 with reduced (61.6 mM) NaCl increased (P < 0.05) the size of the PVS (6.4 µm vs. 2.8 µm) compared to control oocytes that were matured in normal PZM-3 containing 108 mM NaCl. Moreover, oocytes with a larger PVS showed higher CDK1, PCNA, and ERK2 mRNA and intracellular GSH levels (1.6 pixels/oocyte vs. 1.2 pixels/oocyte) and increased blastocyst formation after PA (52.1% vs. 40.6%) and SCNT (31.8% vs. 18.2%) than control oocytes. Our results demonstrate that pig oocytes with a large PVS have greater developmental competence after PA and SCNT, which is attributed to improved cytoplasmic maturation based on the enhanced GSH level and transcription factor expression. Further, enlargement of the PVS by culturing in low-NaCl medium improves the developmental competence of pig oocytes.

Myo-inositol improves the viability of boar sperm during liquid storage.

Introduction: Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Methods: Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Results: Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. Conclusion: The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.

Neurotrophin-4 promotes the specification of trophectoderm lineage after parthenogenetic activation and enhances porcine early embryonic development.

Neurotrophin-4 (NT-4), a neurotrophic factor, appears to affect early embryonic development because it is secreted not only by neurons but also by oviductal and uterine epithelial cells. However, no studies have characterized the effects of NT-4 on early embryonic development in pigs. In this study, we applied the experimental model of parthenogenetic-activation (PA)-derived embryos. Herein, we investigated the effect of NT-4 supplementation during the in vitro culture (IVC) of embryos, analyzed the transcription levels of specific genes, and outlined the first cell lineage specification for porcine PA-derived blastocysts. We confirmed that NT-4 and its receptor proteins were localized in both the inner cell mass (ICM) and trophectoderm (TE) in porcine blastocysts. Across different concentrations (0, 1, 10, and 100 ng/mL) of NT-4 supplementation, the optimal concentration of NT-4 to improve the developmental competence of porcine parthenotes was 10 ng/mL. NT-4 supplementation during porcine IVC significantly (p < 0.05) increased the proportion of TE cells by inducing the transcription of TE lineage markers (CDX2, PPAG3, and GATA3 transcripts). NT-4 also reduced blastocyst apoptosis by regulating the transcription of apoptosis-related genes (BAX and BCL2L1 transcripts) and improved blastocyst quality via the interaction of neurotrophin-, Hippo-yes-associated protein (Hippo-YAP) and mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway. Additionally, NT-4 supplementation during IVC significantly (p < 0.05) increased YAP1 transcript levels and significantly (p < 0.01) decreased LATS2 transcript levels, respectively, in the porcine PA-derived blastocysts. We also confirmed through fluorescence intensity that the YAP1 protein was significantly (p < 0.001) increased in the NT-4-treated blastocysts compared with that in the control. NT-4 also promoted differentiation into the TE lineage rather than into the ICM lineage during porcine early embryonic development. In conclusion, 10 ng/mL NT-4 supplementation enhanced blastocyst quality by regulating the apoptosis- and TE lineage specification-related genes and interacting with neurotrophin-, Hippo-YAP-, and MAPK/ERK signaling pathway during porcine in vitro embryo development.

Tetraploid embryo aggregation produces high-quality blastocysts with an increased trophectoderm in pigs.

Tetraploid complementation is an ideal method for demonstrating the differentiation potential of pluripotent stem cells. In this study, we selected the most efficient tetraploid production method for porcine embryos and investigated whether tetraploid blastomere aggregation could enhance the quality of tetraploid embryos. Three methods were investigated to produce tetraploid embryos: First, tetraploid embryos were produced using electro-fusion of two-cell stage parthenogenetic blastomere (FUTP). Second, somatic cell was injected into the mature oocyte and fused to produce tetraploid embryos. Third, oocytes were matured with Cytochalasin B (CB) for the late 22 h of in vitro maturation to inhibit the first polar body (PB1). Following that, non-PB1 oocytes were treated with CB for 4 h after parthenogenetic activation. There was no significant difference in the blastocyst development rate and tetraploid production rate of the embryos produced through the three methods. However, FUTP-derived blastocysts had a significantly lower percentage of apoptotic cells compared to other methods. The developmental competence of embryos, expression of trophectoderm cell marker genes, and distribution of YAP1 protein were investigated in tetraploid embryos produced using the FUTP method. The FUTP method most effectively prevented apoptosis during porcine tetraploid embryo formation. Tetraploid aggregation-derived blastocysts have a high proportion of trophectoderm with increased expression of the CDX2 mRNA and high YAP1 intensity. High-quality blastocysts derived from a tetraploid embryo aggregation can serve as suitable source material for testing the differentiation potential of pluripotent stem cells for blastocyst complementation in pigs.

Supplementation with fibroblast growth factor 7 during in vitro maturation of porcine cumulus-oocyte complexes improves oocyte maturation and early embryonic development.

In vitro generation of porcine embryos is an indispensable method in the realms of both agriculture and biomedicine. Nonetheless, the extant procedures encounter substantial obstacles pertaining to both the caliber and efficacy of the produced embryos, necessitating extensive research to in vitro maturation (IVM), the seminal commencement phase. One potentially fruitful approach may lie in refining the media and supplements composition utilized for oocyte maturation. Fibroblast growth factor-7 (FGF7), alternatively termed keratinocyte growth factor, is a theca-derived cytokine integral to folliculogenesis. This study aimed to examine the ramifications of supplementing FGF7 during the IVM phase. To determine the FGF7 location and its receptor in porcine ovaries, immunohistochemistry was executed based on follicle size categories (1-2, 3-6, and 7-9 mm). Regardless of follicle size, it was determined that FGF7 was expressed in theca and granulosa cells (GCs), whereas the FGF7 receptor was only expressed in the GCs of the larger follicles. During the IVM process, the maturation medium was supplied with various concentrations of FGF7, aiming to mature porcine cumulus-oocyte complexes (COCs). The data indicated a significant augmentation in the nuclear maturation rate only within the group treated with 10 ng/mL of FGF7 (p < 0.05). Post-IVM, the oocytes diameter exhibited a significant expansion in all groups that received FGF7 supplementation (p < 0.05). Additionally, all FGF7-supplemented groups exhibited a substantial elevation in intracellular glutathione levels, coupled with a noticeable reduction in reactive oxygen species levels (p < 0.05). With respect to gene expressions related to apoptosis, FGF7 treatment elicited a downregulation of pro-apoptotic genes and an upregulation of anti-apoptotic genes. The expression of genes associated with antioxidants underwent a significant enhancement (p < 0.05). In terms of the FGF7 signaling pathway-associated genes, there was a significant elevation in the mRNA expression of ERK1, ERK2, c-kit, and KITLG (p < 0.05). Remarkably, the group of 10 ng/mL of FGF7 demonstrated an appreciable uptick in the blastocyst formation rate during embryonic development post-parthenogenetic activation (p < 0.05). In conclusion, the FGF7 supplementation during IVM substantially augments the quality of matured oocytes and facilitates the subsequent development of parthenogenetically activated embryos. These results offer fresh perspectives on improved maturation and following in vitro evolution of porcine oocytes.

Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs.

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

The effect of C-C motif chemokine ligand 2 supplementation on in vitro maturation of porcine cumulus-oocyte complexes and subsequent developmental competence after parthenogenetic activation.

Porcine embryos are used for a variety of applications. However, the maturation rate in vitro remains low, and novel in vitro maturation (IVM) techniques that facilitate the collection of mature oocytes are necessary. C-C motif chemokine ligand 2 (CCL2) is a key periovulatory chemokine present in cumulus-oocyte complexes (COCs). We aimed to examine the effects of CCL2 supplementation during IVM on oocyte maturation and embryonic development. The CCL2 concentration was significantly higher in porcine follicular fluid (pFF) derived from follicles >8 mm in size than in pFF derived from smaller follicles. There was a significant increase in CCL2 mRNA levels in all follicular cells after IVM compared with that before IVM. We analyzed the localization of CCL2 and its receptor, the CCL2 receptor, in follicular cells. During IVM, different concentrations of CCL2 were added to COCs cultured in a maturation medium. After IVM, the group treated with 100 ng/mL CCL2 showed significantly higher metaphase II rates than the control group. All CCL2-treatment groups showed a significant increase in intracellular glutathione levels and a significant decrease in reactive oxygen species levels, compared to the control. In CCs treated with 100 ng/mL CCL2, the mRNA levels of BAX, CASP3, and NPR2 were significantly decreased. Furthermore, the mRNA levels of SOD1, SOD2, and CD44 were significantly increased. In oocytes treated with 10 ng/mL CCL2, mRNA levels of BAX and CASP3 were significantly decreased, whereas, NRF2 and NPM2 were significantly increased. ERK1 exhibited significantly increased mRNA expression in both CCs and oocytes treated with 10 ng/mL CCL2. The protein expression ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly increased in CCs treated with 10 ng/mL CCL2. After parthenogenetic activation, cleavage rates were significantly improved in the 100 ng/mL CCL2 treatment group, and blastocyst formation rates were significantly enhanced in the 10 ng/mL CCL2 treatment group. Overall, our results suggest that IVM medium along with CCL2 improves porcine oocyte maturation and the development of parthenogenetically-activated embryos.