In vitro culture (IVC) can be used for a variety of assisted reproductive technologies. However, IVC in dog has been low efficient compared to other mammalian. It is believed that an embryo developmental block in IVC embryos is cause of low production efficiency. There is no study of embryo developmental block in dog yet. In this study, we attempted to estimate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, ROS activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Moreover, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Furthermore, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, these results indicated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

Generally, in vivo, primary oocytes are grown and matured into secondary oocytes in the ovarian follicles. Quality of the oocytes matured in vivo is higher than that of oocytes matured in vitro, indicating the importance of materializing the microenvironment of ovarian follicles for production of high quality oocyte. Therefore, we tried to mimic the stiffness of ovarian follicles using an agarose as a biocompatible natural polymer. Unfortunately, to date, there are no many reports on whether the quality of porcine oocytes can be increased effectively under the soft matrix. Accordingly, we tried to evaluate the effects of IVM using different mechanical properties of agarose substrate on developmental competence of porcine oocytes. Agarose substrate was constructed and cumulus-oocyte-complexes (COCs) retrieved from porcine medium antral follicles were matured on non-coated (control) culture dish or dishes coated with 1% and 2% (w/v) agarose substrate. Then, cumulus expansion, embryonic development after parthenogenetic activation, and gene expression level were analyzed and compared. As the results, significant increase in blastocyst formation and cumulus expansion were detected in COCs matured on 1% (w/v) agarose substrate compared with control. Moreover, oocytes of COCs matured on 1% (w/v) agarose substrate showed significantly higher BMP15 expression level compared with control. Pro-apoptotic gene BAX expression was significantly increased in oocytes of COCs matured on 2% (w/v) agarose substrate compared with control. In the glycolytic enzyme phosphofructokinase (PFKP) gene expression, cumulus cells of COCs matured on agarose substrate showed significantly higher PFKP expression than control while they showed significantly lower BAX expression than control. These results demonstrated that quality of porcine oocytes could be increased efficiently by the IVM of immature oocytes on the soft culture matrix using agarose.

This study was conducted to evaluate the effects of insulin and epidermal growth factor (EGF) in a in vitro growth (IVG) medium on oocyte growth, in vitro maturation (IVM) and embryonic development of pig oocytes derived from small antral follicles (SAF) less than 3 mm in diameter. SAF oocytes were cultured for 2 days to induce IVG in alpha-minimal essential medium supplemented with 1 mM dbcAMP and 15% (v/v) fetal bovine serum. After IVG culture, oocyte maturation was induced by culturing IVG oocytes in IVM medium for 44 h. IVM oocytes that extruded the first polar body were selected and induced for parthenogenesis (PA) by applying electric stimulus. SAF oocytes cultured under the insulin treatment showed a significantly increased (P < 0.05) nuclear maturation (73.8%) compared to those cultured with insulin and EGF (59.8%). After PA, the proportions of blastocysts based on the number of metaphase II oocytes were significantly higher (P < 0.05) in oocytes that were cultured for IVG with insulin, EGF, and insulin + EGF (32.4%, 35.2%, and 34.8%, respectively) than in control (22.9%). IVG oocytes treated with insulin showed an increased oocyte diameter (116.3 μm) compared to those treated with insulin and EGF (114.0 μm) (P < 0.05). Intra-oocyte GSH content significantly increased (1.07 pixels/oocyte) by insulin treatment during IVG compared to that of oocytes treated with insulin + EGF (0.78 pixels/oocyte). These results demonstrate that IVG culture of SAF oocytes under insulin or/and EGF treatment supports oocyte maturation and improves embryonic development to the blastocyst stage after PA in pigs.

U0126 is a highly selective inhibitor of both MEK1 and MEK2, a type of MAPK/ERK kinase. This study was conducted to evaluate the effect of U0126 treatment during in vitro maturation (IVM) on nuclear maturation, intra-oocyte glutathione content, and embryonic development after parthenogenesis (PA). U0126 (5 μM) was supplemented to IVM medium during the first 0 (control), 2, and 4 h. The basic medium used for IVM was medium-199 supplemented with 10% (v/v) porcine follicular fluid (standard), 0.6 mM cysteine, 0.91 mM pyruvate, 75 μg/ml kanamycin, and 1 μg/ml insulin. Immature pig oocytes were matured for 44 h and then oocytes reached metaphase II stage were electrically activated to induce PA. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) fatty acid-free BSA. When immature oocytes were treated with U0126 during the first 0, 2, 4 h of IVM culture, nuclear maturation was significantly (P < 0.05) increased by the U0126 treatment for 4 h (96.2 ± 1.3%) compared to standard IVM (90.6 ± 2.1%). Cleavage of PA embryos was significantly increased by 4 h- treatment (90.6 ± 2.2%) compared to standard medium (83.9 ± 1.8%). In addition, blastocyst formation of PA embryos was significantly (P < 0.05) increased by the treatment for 4 h (55.8 ± 5.7%) compared to 2 h (38.1 ± 6.1%). The glutathione contents in IVM oocytes were not altered by the U0126 treatments for 0, 2, and 4 h (1.28 ± 0.10, 1.16 ± 0.09, and 1.10 ± 0.09, respectively). Our results demonstrated that 5 μM U0126 treatment during the first 4 h of IVM showed positive effects on nuclear maturation, cleavage, and embryonic development in pigs.

Microenvironments surrounded with various extracellular matrix (ECM) components can decide specifically the fate of spermatogonial stem cells (SSCs) and integrin heterodimers recognizing directly ECM proteins play an important role in transporting ECM-derived signals into cytoplasm, resulting in inducing a variety of biological functions such as cell attachment, self-renewal and differentiation. However, to date, studies on type of integrin heterodimers expressed functionally on the undifferentiated SSCs derived from mouse with hybrid strain remain unclear. Therefore, we tried to investigate systematically what kind of integrin heterodimers are expressed transcriptionally, translationally and functionally in the SSCs derived from testis of hybrid (B6CBAF1) mouse. For these, magnetic activated cell sorting (MACS) using Thy1 antibody was used for isolating SSCs from testis, and real-time PCR or fluorescence immunoassay was conducted for measuring transcriptional or translational level of integrin α and β subunits in the isolated SSCs. Subsequently, antibody inhibition assay was conducted for confirming functionality of presumed integrin heterodimers. As the results, transcriptional levels of genes encoding total 25 integrin subunits were quantified, 7 integrin α (α4, α6, α7, α9, αV, αL and αE) and 2 integrin β (β1 and β5) subunit genes showed significantly increased transcriptional up-regulation, compared to the other integrin subunit genes. In contrast, integrin α3, α5, α10 and α11, and integrin β2, β3, β4 and β7 were weakly transcribed. When translational levels of the integrin α subunits showing high transcription level (α4, α6, α7, α9, αV, αL and αE) were measured, significantly strong translational up-regulation of integrin α6, α7, α9, αV and αL subunit genes were detected, whereas integrin α4 and αE subunit genes were weakly. In case of integrin β subunit, β1 evaluated more expression than β5. Based on these results, we speculated that the undifferentiated SSCs derived from B6CBAF1 mouse might express integrin α4β 1, α6β1, α7β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Subsequently, the hybrid strain SSCs showed significantly increased adhesion to fibronectin, laminin, tenascine-C and vitronectin and functional blocking of integrin α4β1, α6β1, α9β1, and αVβ1 or αVβ5 in SSCs significantly inhibited attachment to fibronectin, laminin, tenascin-C and vitronectin, respectively. Accordingly, we could identify that the hybrid (B6CBAF1) mouse-derived SSCs had integrin α4β1, α6β1, α9β1, αVβ1 or αVβ5 on plasma membrane. Moreover, this information will greatly contribute to constructing non-cellular niche supporting self-renewal of SSCs in the future.

Poor embryo quality and low blastocyst formation have been major limitations in establishment of cloned embryonic stem cells and production of cloned animals through somatic cell nuclear transfer (SCNT). Aggregation of embryos is a promising method for improving developmental competence of blastocysts. The aim of this study was to improve the blastocyst formation and the quality of parthenogenetic (PA) pig embryos by the aggregation of blastomeres at the 4-cell stage that were cultured in various type of culture dishes with or without phytohemagglutinin (PHA). The PA embryos were produced by the general method of our laboratory. On Day 2 after PA, the zona pellucida of 4 cell-stage embryos were removed by treatment with 0.5% (wt/vol) pronase solution. The 3x zona-free blastomere (ZFB) were randomly distributed in each of the following treatments for aggregation. ZFB were cultured for 5 days at 39℃ in an atmosphere 5% CO2, 5% O2, and 90% N2. In Experiment 1, effect of culture dishes on the aggregation efficiency and developmental competence of PA embryos were investigated. ZFB were cultured on non-coated (control) culture dish or dishes coated with 1% (wt/vol) agarose substrate (AS) or Well of the Well in dishes coated with 1% (wt/vol) agarose substrate (WAS). The ZFB cultured in WAS showed significantly higher (P<0.05) aggregation (81.2%) than AS and control (21.6-45.5%). The mean cell number in blastocysts derived from AS and WAS (81.4-89.3 cells/blastocyst) was significantly higher (P<0.05) than that of control (63.8 cells/blastocyst). In Experiment 2, effects of 150 ug/ml PHA treatment on the aggregation efficiency and developmental competence of embryos were investigated. The ZFB cultured in AS with PHA showed a higher (P<0.05) aggregation rate (90.0%) than that in AS without PHA, control with PHA, and control (39.2%, 57.9% and 17.5%, respectively). In conclusion, aggregation of porcine ZFB treated with PHA and agarose substrate could be a useful technique for producing improving blastocyst development with increased mean cell number of blastocysts in pigs.