Rapamycin rescues the poor developmental capacity of aged porcine oocytes.

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 µM rapamycin/24 h, 47.52±5.68) or control oocytes (44 h IVM; 42.14±4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos.

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

Investigation of the Developmental Potential and Developmental Kinetics of Bovine Parthenogenetic and Somatic Cell Nuclear Transfer Embryos Using a Time-Lapse Monitoring System.

A time-lapse monitoring system has predictive value for selecting good-quality embryos with the highest implantation potential. Using this new tool, we investigated the developmental potential and developmental kinetics of bovine parthenogenetic (PA) and two types of somatic cell nuclear transfer (NT) embryos. Bovine non-transgenic ear cells (bECs) or transgenic cells (bTGCs) were used as donor cells. The cleavage and blastocyst development rates did not significantly differ among the PA, NT-bEC, and NT-bTGC groups, and first cleavage occurred an average of 19.3 hours (n = 70), 21.6 hours (n = 60), and 21.3 hours (n = 62) after activation, respectively (20.4 hours [n = 192] for all embryos). When embryos were classified into early cleaving (≤20 hours) and late cleaving (>20 hours) groups, the blastocyst formation rate was much higher in the early cleaving groups (PA, 46%; NT-bEC, 50%; NT-bTGC, 39%) than in the late cleaving groups (PA, 18%; NT-bEC, 23%; NT-bTGC, 28%), while the percentage of embryos whose development was blocked between the two- and eight-cell stages was increased in the late cleaving groups. The percentage of embryos classified as early cleaving with a normal morphology was twofold higher in the PA group (20.0%, n = 14) than in the NT-bTGC group (9.7%, n = 6). The timing of each developmental stage varied widely; the timing of first cleavage varied from 7.6 hours in the PA group to 34.5 hours in the NT-bEC group and the timing of expanded/hatching blastocyst appearance varied from 141.6 hours in the PA group to 196.3 hours in the NT-bTGC group, differences of 26.9 and 54.7 hours, respectively (PA>NT-bEC>NT-bTGC). These results demonstrate that time-lapse monitoring provides novel data regarding individual embryo developmental kinetics and helps to predict developmental potential for improved bovine NT embryo selection based on early cleavage (≤20 hours) and normal morphology.

Effective Oocyte Vitrification and Survival Techniques for Bovine Somatic Cell Nuclear Transfer.

Bovine somatic cell nuclear transfer (SCNT) using vitrified-thawed (VT) oocytes has been studied; however, the cloning efficiency of these oocytes is not comparable with that of nonvitrified (non-V) fresh oocytes. This study sought to optimize the survival and cryopreservation of VT oocytes for SCNT. Co-culture with feeder cells that had been preincubated for 15 h significantly improved the survival of VT oocytes and their in vitro developmental potential following SCNT in comparison to co-culture with feeder cells that had been preincubated for 2, 5, or 24 h (p<0.05). Spindle assessment via the Oosight Microscopy Imaging System and microtubule staining revealed that vitrified metaphase II oocytes (VT group) were not suitable for SCNT. However, enucleating and/or activating oocytes prior to freezing enhanced their developmental potential and suitability for SCNT. The cloning efficiency of the enucleated-activated-vitrified-thawed (EAVT) group (21.6%) was better than that of the other vitrification groups [enucleated-vitrified-thawed (EVT) group, 13.7%; VT group, 15.0%; p<0.05] and was comparable with that of the non-V group (25.9%). The reactive oxygen species level was significantly lower in the EAVT group than in the other vitrification groups (p<0.05). mRNA levels of maternal genes (ZAR1, BMP15, and NLRP5) and a stress gene (HSF1) were lower in the vitrification groups than in the non-V group (p<0.05), whereas the level of phospho-p44/42 mitogen-activated protein kinase did not differ among the groups. Among the vitrification groups, blastocysts in the EAVT group had the best developmental potential, as judged by their high mRNA expression of developmental potential-related genes (POU5f1, Interferon-tau, and SLC2A5) and their low expression of proapoptotic (CASP3) and stress (Hsp70) genes. This study demonstrates that SCNT using bovine frozen-thawed oocytes can be successfully achieved using optimized vitrification and co-culture techniques.

Establishment of bovine embryonic stem cell lines using a minimized feeder cell drop.

Bovine embryonic stem cells (ESCs) are a powerful tool for agricultural and biomedical applications. The purpose of this study was to introduce a new method for generating bovine ESCs. Mechanically isolated bovine inner cell masses (ICMs) from in vitro-produced blastocysts were cultured individually on a 10-µL mouse embryonic fibroblast (MEF) feeder cell drop covered with oil. From 126 blastocysts classified by their developmental stage and ICM size, 21 primary bovine ESC-like colonies were formed (16.7%) and established six JNU (Jeju National University)-ibES cell lines (28.6%, 6/21; hatched blastocyst×4, hatching blastocyst×1, and expanded blastocyst×1). These cells exhibited typical ESC morphology, and pluripotency markers were detected through immunocytochemistry, RT-PCR, and real-time RT-PCR, including Oct4, stage-specific embryonic antigen-1 (SSEA-1), Nanog, Tumor rejection antigen-1-81, Rex1, and alkaline phosphatase. Through RT-PCR analysis of spontaneous differentiation, gene expression of all three embryonic germ layers was detected: ectodermal (Pax6 and DBH), mesodermal (CMP and Enolase), and endodermal [alpha fetoprotein (α-FP) and albumin]. In addition, JNU-ibES cell lines were directed differentiated into neuronal (Map2 and Tuj1) and glial (GFAP) cells. Bovine ESC lines had a normal karyotype, with a chromosome count of 58+XY (JNU-ibES-05). This is the first trial investigating a minimized microdrop culture method for the generation of bovine ESCs. These results demonstrated that the minimized MEF feeder cell drop can support the establishment of bovine ESC lines.

Improved cloning efficiency and developmental potential in bovine somatic cell nuclear transfer with the oosight imaging system.

In somatic cell nuclear transfer (SCNT) procedures, exquisite enucleation of the recipient oocyte is critical to cloning efficiency. The purpose of this study was to compare the effects of two enucleation systems, Hoechst staining and UV irradiation (hereafter, irradiation group) and Oosight imaging (hereafter, Oosight group), on the in vitro production of bovine SCNT embryos. In the Oosight group, the apoptotic index (2.8 ± 0.5 vs. 7.3 ± 1.2) was lower, and the fusion rate (75.6% vs. 62.9%), cleavage rate (78.0% vs. 63.7%), blastocyst rate (40.2% vs. 29.2%), and total cell number (128.3±4.8 vs. 112.2 ± 7.6) were higher than those in the irradiation group (all p<0.05). The overall efficiency after SCNT was twice as high in the Oosight group as that in the irradiation group (p<0.05). The relative mRNA expression levels of Oct4, Nanog, Interferon-tau, and Dnmt3A were higher and those of Caspase-3 and Hsp70 were lower in the Oosight group compared with the irradiation group (p<0.05). This is the first report to show the positive effect of the Oosight imaging system on molecular gene expression in the SCNT embryo. The Oosight imaging system may become the preferred choice for enucleation because it is less detrimental to the developmental potential of bovine SCNT embryos.