Protective immune response induced by Leghorn male hepatoma cell-adapted fowl adenovirus-4.

Fowl adenovirus-4 (FAdV-4) is a highly contagious virus that causes acute and lethal hepatitis. It leads to substantial economic losses in the poultry industry. Among the structural proteins of FAdV-4, hexon and fiber2 are associated with immunopathogenesis. A frameshift mutation was generated in the fiber2 protein by seral passages in the Leghorn male hepatoma (LMH) cell line. Immunization using the attenuated virus (80 times passaged) before the virulent FAdV-4 challenge protected hosts from the infection and cleared the invading virus. In immunized animals, activated CD4+ and CD8+ T cell populations were larger during the FAdV-4 challenge. The change in the B cell population was similar. Myeloid cells were highly increased during FAdV-4 infection after the immunization, but the immunization inhibited the expansion in both liver and spleen. The functional gene expression for immune modulation was strongly associated with immune cell changes in the liver, however, this association was not strong in the spleen. The present findings imply that genetic modification by cellular adaptation regulates immune cell phenotype and function in the target organ. In addition, we suggest the attenuated virus as a protective strategy against the novel FAdV-4 strains.

Deep Learning-Based Precision Analysis for Acrosome Reaction by Modification of Plasma Membrane in Boar Sperm.

The analysis of AR is widely used to detect loss of acrosome in sperm, but the subjective decisions of experts affect the accuracy of the examination. Therefore, we develop an ARCS for objectivity and consistency of analysis using convolutional neural networks (CNNs) trained with various magnification images. Our models were trained on 215 microscopic images at 400× and 438 images at 1000× magnification using the ResNet 50 and Inception-ResNet v2 architectures. These models distinctly recognized micro-changes in the PM of AR sperms. Moreover, the Inception-ResNet v2-based ARCS achieved a mean average precision of over 97%. Our system's calculation of the AR ratio on the test dataset produced results similar to the work of the three experts and could do so more quickly. Our model streamlines sperm detection and AR status determination using a CNN-based approach, replacing laborious tasks and expert assessments. The ARCS offers consistent AR sperm detection, reduced human error, and decreased working time. In conclusion, our study suggests the feasibility and benefits of using a sperm diagnosis artificial intelligence assistance system in routine practice scenarios.

Effect of Magnetized Freezing Extender on Membrane Damages, Motility, and Fertility of Boar Sperm Following Cryopreservation.

Magnetized water is defined as the amount of water that has passed through a magnet. The magnetic field weakens the hydrogen bonds between the water molecules, leading to the magnetized liquid acquiring special characteristics such as easy supercooling and forming smaller ice crystals. We researched the influences of a magnetized freezing extender on cell membrane damage and in vitro fertilization of boar sperm during cryopreservation. The freezing extenders were passed through 0, 2000, 4000, and 6000 gausses (G) of magnetic devices using a liquid cycling pump system and then used for the sperm freezing process. The damage to plasma, acrosomal, and mitochondrial membranes in frozen-thawed spermatozoa was investigated by flow cytometry, and motility was assessed using the CASA system. The fertility of frozen-thawed sperm was estimated using in vitro fertilization. The damage to the membranes was significantly decreased in the magnetized freezing extender by the 6000 G magnetic field compared to that of the control in frozen-thawed sperm, and motility was increased in the 6000 G group. Although there were no significant differences in the cleavage rates of in vitro fertilized oocytes among the treatment groups, the ratio of blastocyst formation increased in the magnetized freezing extender groups compared with that in the control group. The number of blastocysts was significantly higher in the 4000 G group than in the 0 G group. In conclusion, these results suggest that a magnetized freezing extender could improve the freezability of sperm and the development of oocytes fertilized in vitro with frozen-thawed sperm.

5-Azacytidine (5-aza) Induces p53-associated Cell Death Through Inhibition of DNA Methyltransferase Activity in Hep3B and HT-29 Cells.

BACKGROUND/AIM: DNA methylation regulates the expression of genes that control mechanisms of cell death. TP53 gene expression inhibits tumorigenesis, and its action is closely associated with cell death. 5-Azacytidine (5-aza), increases the expression of the TP53 gene by inhibiting DNA methyltransferase. MATERIALS AND METHODS: Using 5-aza, we induced DNA hypomethylation in p53-null and p53-expressing cancer cell lines and investigated potential mechanisms of cancer cell death. RESULTS: TP53 expression promoted cell death. Notably, methylation-specific PCR (MSP) and bisulfite sequencing revealed more methylation sites at the TP53 promoter region in p53-null cells than in p53-expressing cells. CONCLUSION: This study suggests a novel mechanism of tumorigenesis regulated by p53 expression.

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor.

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 µM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

Effect of 5-azacytidine (5-aza) on UCP2 expression in human liver and colon cancer cells.

The function of the uncoupling protein 2 (UCP2) is different for each cancer cell. However, the mechanism of expression is still unclear. DNA methylation affects protein expression and is one factor that transforms normal cells into cancer cells. In this study, the hepatocellular carcinoma Hep3B and HepG2 cells and colorectal cancer HT-29 cells were treated with 5-azacytidine (5-aza), a DNA demethylation agent, to observe the modification of UCP2 expression and the methylation degree in the UCP2 promoter region. Promoter basal activity and degree of UCP2 expression were measured in Hep3B, HepG2, and HT-29 cells. In addition, methylation-specific PCR (MSP) was performed to investigate the degree of methylation in the UCP2 promoter region. The methylation region in the UCP2 promoter was confirmed based on bisulfite sequencing. In Hep3B cells in which UCP2 mRNA was not transcribed, the promoter basal activity was significantly higher than in HT-29 or HepG2 cells in which UCP2 mRNA was transcribed. Treatment with 5-aza increased UCP2 expression in Hep3B and HT-29 cells; however, the expression in HepG2 cells was unchanged. The UCP2 promoter in Hep3B cells has numerous methylated regions compared with HT-29 and HepG2 cells. The results of the present study revealed that inhibition of UCP2 expression in Hep3B cells was due to methylation of the promoter region. Investigating the mechanism that induces UCP2 expression in cancer cells is important to understand the function of UCP2, which could aid in cancer treatment.

Endoplasmic Reticulum (ER) Stress Inhibitor or Antioxidant Treatments during Micromanipulation Can Inhibit Both ER and Oxidative Stresses in Porcine SCNT Embryos.

We investigated the effects of endoplasmic reticulum (ER) stress inhibitor and antioxidant treatments during the micromanipulation of somatic cell nuclear transfer (SCNT) on in vitro development of SCNT embryos. Tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor and vitamin C (Vit. C), an antioxidant, were treated by alone or in combination, then, the level of X-box binding protein 1 (Xbp1) splicing and the expressions of ER stress-associated genes, oxidative stress-related genes, and apoptotic genes were confirmed in the 1-cell and blastocyst stages. In the 1-cell stage, the levels of Xbp1 splicing were significantly decreased in TUDCA and Vit. C treatment groups compared to the control (p<0.05). In addition, the expression levels of most ER stress-associated genes and oxidative stress-related genes were significantly lower in all treatment groups than the control (p<0.05), and the transcript levels of apoptotic genes were also significantly lower in all treatment groups than the control (p<0.05). In the blastocyst stage, decreased expression of ER stress-, oxidative stress-, and apoptosis-related genes were observed only in some treatments. However, the blastocyst formation rates in TUDCA and Vit. C treatment groups (24.8% and 22.0%, respectively) and mean blastocyst cell number in all treatment groups (59.7±4.3 to 63.5±3.3) were significantly higher (p<0.05) than those of control. The results showed that the TUDCA or Vit. C treatment during micromanipulation inhibited both ER and oxidative stresses in the early stage of SCNT embryos, thereby reducing cell damage and promoting in vitro development.

Effect of Alpha-Linolenic Acid with Bovine Serum Albumin or Methyl-Beta-Cyclodextrin on Membrane Integrity and Oxidative Stress of Frozen-Thawed Boar Sperm.

The study was conducted to investigate the effects of alpha-linolenic acid (ALA) combined with bovine serum albumin (BSA) or methyl-beta-cyclodextrin (MBCD) on plasma and acrosomal membrane damages, mitochondrial activity, morphological abnormality, motility, and oxidative stress in frozen-thawed boar sperm. In previous our study, 3 ng/mL ALA had been shown protective effect during freezing process of boar sperm. Therefore, we used 3 ng/mL ALA in present study and ALA was combined with same molar ratio of BSA or MBCD (ALA+BSA and ALA+MBCD, respectively). To confirm the effect of two carrier proteins, same volume of BSA and MBCD without ALA were added during cryopreservation. Membrane damage, mitochondrial activity, reactive oxygen species (ROS) and lipid peroxidation (LPO) levels were measured using flow cytometry, and movement of sperm tail as motility parameter and morphological abnormality were observed under light microscope. In results, all of sperm parameters were enhanced by ALA combined with BSA or MBCD compared to control groups (p<0.05). Mitochondrial activity, morphological abnormality, ROS and LPO levels in ALA+BSA or MBCD groups were no significant difference compared with ALA, BSA and MBCD treatment groups. On the other hand, plasma and acrosomal membrane intact, and sperm motility in ALA+MBCD group were higher than single treatment groups (p<0.05), whereas ALA+BSA did not differ. Our findings indicate that carrier proteins such as BSA and MBCD could improve the effect of ALA during cryopreservation of boar sperm, and treatment of ALA with carrier proteins enhance membrane integrity, mitochondrial activity through reduction of ROS-induced LPO.

Effects of Endoplasmic Reticulum Stress Inhibitor Treatment during the Micromanipulation of Somatic Cell Nuclear Transfer in Porcine Oocytes.

We examined the effects of endoplasmic reticulum (ER) stress inhibitor treatment during the micromanipulation of porcine somatic cell nuclear transfer (SCNT) on the in vitro development of SCNT embryos. ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxycholic acid (TUDCA; 100 µM) were added to the micromanipulation medium and holding medium. The expression of X-box binding protein 1 (Xbp1), ER-stress-associated genes, and apoptotic genes in SCNT embryos was confirmed at the one-cell and blastocyst stages. Levels of Xbp1 splicing and expression of ER-stress-associated genes in SCNT embryos at the one-cell stage decreased significantly with TUDCA treatment (p<0.05). The expression of ER-stress-associated genes also decreased slightly with the addition of both salubrinal and TUDCA (Sal+TUD). The expression levels of caspase-3 and Bcl2-associated Xprotein (Bax) mRNA were also significantly lower in the TUDCA and Sal+TUD treatments (p<0.05). At the blastocyst stage, there were no differences in levels of Xbp1 splicing, and transcription of ER-stress-associated genes and apoptosis genes between control and treatment groups. However, the blastocyst formation rate (20.2%) and mean blastocyst cell number (63.0±7.2) were significantly higher (p<0.05) for embryos in the TUDCA treatment compared with those for control (12.6% and 41.7±3.1, respectively). These results indicate that the addition of ER-stress inhibitors, especially TUDCA, during micromanipulation can inhibit cellular damage and enhance in vitro development of SCNT embryos by reducing stress levels in the ER.

Correlation of spontaneous adipocyte generation with osteogenic differentiation of porcine skin-derived stem cells.

The objective of this study was to examine effects of spontaneous adipocyte generation on osteogenic differentiation of porcine skin-derived stem cells (pSSCs). Correlation between osteogenic differentiation and adipocyte differentiation induced by osteocyte induction culture was determined using different cell lines. Osteogenic differentiation efficiency of pSSCs was then analyzed by controlling the expression of adipocyte-specific transcription factors during osteogenic induction culture. Among four cell lines, pSSCs-II had the lowest lipid droplet level but the highest calcium content (p ＜ 0.05). It also expressed significantly low levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2) and adipocyte protein 2 (aP2) mRNAs but very high levels of runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) mRNAs as osteogenic makers (p ＜ 0.05). Oil red O extraction was increased by 0.1 µM troglitazone (TGZ) treatment but decreased by 50 µM bisphenol A diglycidyl ether (BADGE) (p ＜ 0.05). Calcium content was drastically increased after BADGE treatment compared to that in osteogenic induction control and TGZ-treated pSSCs (p ＜ 0.05). Relative expression levels of PPARγ2 and aP2 mRNAs were increased by TGZ but decreased by BADGE. Expression levels of Rucx2 and ALP mRNAs, osteoblast-specific marker genes, were significantly increased by BADGE treatment (p ＜ 0.05). The expression level of BCL2 like 1 was significantly higher in BADGE-treated pSSCs than that in TGZ-treated ones (p ＜ 0.05). The results demonstrate that spontaneous adipocyte generation does not adversely affect osteogenic differentiation. However, reducing spontaneous adipocyte generation by inhibiting PPARγ2 mRNA expression can enhance in vitro osteogenic differentiation of pSSCs.

Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos.

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) E+10 µM Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 µM) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

Effects of 17ß-estradiol, Interleukin-1ß, and Human Chorionic Gonadotropin on Activity and mRNA Expression of Plasminogen Activators in Porcine Endometrial Cells.

This study aimed to investigate changes in the activity and mRNA expression of plasminogen activators (PAs) induced by 17ß-estradiol (E2), human chorionic gonadotropin (hCG), and interleukin-1ß (IL-1ß) in porcine endometrial cells. Endometrial cells were isolated from the epithelium and cultured to 80% confluence. They were then treated for 24 h with E2 (0.2, 2, 20, and 200 ng/mL), IL-1ß (0.1, 1, 10, and 100 ng/mL), and hCG (0.5, 1, 1.5 and 2 IU/mL). mRNA expressions of urokinase-type (uPA) and tissue-type (tPA) PAs were analyzed using reverse transcription PCR, and activities were measured using a PA activity assay. mRNA expressions of uPA and tPA increased with E2 treatment; however, this was not significant. Similarly, treatment with hCG did not influence the mRNA expressions of PAs. Interestingly, treatment with 0.1 ng/mL IL-1ß significantly reduced the mRNA expression of uPA, but did not affect that of tPA. Treatment with 2, 20, and 200 ng/mL E2 increased PA activity compared with the control group; treatment with 0.1 and 1 ng/mL IL-1ß significantly increased PA activity compared with the other IL-1ß treatment groups, whereas treatment with 10 and 100 ng/mL IL-1ß decreased. Treatment with 2 IU/mL hCG increased PA activity compared with the other treatment groups, although there were no significant differences between the hCG and control groups. In conclusion, the activity and mRNA expression of PAs were differently regulated by the hormone/cytokine and its concentration in porcine endometrial cells. Therefore, understanding PA regulatory mechanisms may help to improve the reproductive potential of domestic animals.

Analysis of Endoplasmic Reticulum (ER) Stress Induced during Somatic Cell Nuclear Transfer (SCNT) Process in Porcine SCNT Embryos.

This study investigates the endoplasmic reticulum (ER) stress and subsequent apoptosis in duced during somatic cell nuclear transfer (SCNT) process of porcine SCNT embryos. Porcine SCNT and in vitro fertilization (IVF) embryos were sampled at 3 h and 20 h after SCNT or IVF and at the blastocyst stage for mRNA extraction. The x-box binding protein 1 (Xbp1) mRNA and the expressions of ER stress-associated genes were confirmed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. Before commencing SCNT, somatic cells treated with tunicamycin (TM), an ER stress inducer, confirmed the splicing of Xbp1 mRNA and increased expressions of ER stress-associated genes. In all the embryonic stages, the SCNT embryos, when compared with the IVF embryos, showed slightly increased expression of spliced Xbp1 (Xbp1s) mRNA and significantly increased expression of ER stress-associated genes (p<0.05). In all stages, apoptotic gene expression was slightly higher in the SCNT embryos, but not significantly different from that of the IVF embryos except for the Bax/Bcl2L1 ratio in the 1-cell stage (p<0.05). The result of this study indicates that excessive ER stress can be induced by the SCNT process, which induce apoptosis of SCNT embryos.

Alpha-Linolenic Acid: It Contribute Regulation of Fertilization Capacity and Subsequent Development by Promoting of Cumulus Expansion during Maturation.

The objective of this study was to evaluate the effects of alpha-linolenic acid (ALA) during in vitro maturation (IVM) on cumulus expansion, nuclear maturation, fertilization capacity and subsequent development in porcine oocytes. The oocytes were incubated with 0, 25, 50, and 100 µM ALA. Cumulus expansion was measured at 22 h, and gene expresison and nuclear maturation were analyzed at 44 h after maturation. Then, mature oocytes with ALA were inseminated, and fertilization parameters and embryo development were evaluated. In results, both of cumulus expansion and nuclear maturation were increased in 50 µM ALA groups compared to control groups (p<0.05). However, expression of gap junction protein alpha 1 (GJA1, cumulus expansion-related gene), delta-6 desaturase (FADS1, fatty acid metabolism-related gene), and delta-5 desaturase (FADS2) mRNA in cumulus cells were reduced by 50 µM ALA treatment (p<0.05). Cleavage rate was enhanced in 25 and 50 µM ALA groups (p<0.05), especially, treatment of 50 µM ALA promoted early embryo develop to 4 and 8 cell stages (p<0.05). However, blastocyst formation and number of cells in blastocyst were not differ in 25 and 50 µM ALA groups. Our findings show that ALA treatment during maturation could improve nuclear maturation, fertilization, and early embryo development through enhancing of cumulus expansion, however, fatty acid metabolism- and cumulus expansion-related genes were down-regulated. Therefore, addition of ALA during IVM of oocytes could improve fertilization and developmental competence, and further studies regarding with the mechanism of ALA metabolism are needed.

Effects of Progesterone and 17ß-Estradiol under Presence or Absence of FBS on Plasminogen Activators Activity in Porcine Uterine Epithelial Cells.

The present study was conducted to investigate the regulatory mechanism of plasminogen activators (PAs) activation by 17ß-estradiol (E2) and progesterone (P4) in porcine uterine epithelial cells (pUECs). pUECs were collected from porcine uterine horn and cultured at 80% confluence. Then, 0.1% (v/v) DMSO, 20 ng/mL E2, and P4 with or without fetal bovine serum (FBS) treated to cultured cells for 24 hours. The supernatants were used for measurement of PAs activity and expression of urokinase-type PA (uPA), tissue-type PA (tPA), uPA specific receptor (uPAR), and type-1 PA inhibitor (PAI-1) mRNA were analyzed by real-time PCR. The expression of PAs-related genes was not affect by steroid hormones in both of serum treatment groups. However, PAs activity was increased by treatment of E2 compared to 0.1% DMSO treatment in serum-free group (p<0.05). Then, E2 and P4 were diluted with 0.002% (v/v) DMSO for reduction of its effect and treated to cultured cells without FBS. Only tPA mRNA was significantly increased by E2 treatment (p<0.05). PAs activity was enhanced in E2 treated group compared to control groups (p<0.05). These results indicate that serum-free condition is more proper to evaluate effect of steroid hormones and activation of PAs in pUECs was mainly regulated by estrogen. These regulation of PAs activation may be associated with uterine remodeling during pre-ovulatory phase in pigs, however, further studies are needed to investigate precise regulatory mechanism.

Erratum to : Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos.

[This corrects the article DOI: 10.12717/DR.2018.22.3.235.].

Treatment of Epidermal Growth Factor (EGF) enhances Nuclear Maturation of Porcine Oocytes and Stimulates Expression of ER/Golgi Transport Proteins.

This study was conducted to investigate stimulatory effect of epidermal growth factor (EGF) on nuclear maturation and the expression level of EGF-receptor (EGFR), GM-130 (a marker of Golgi apparatus), transport protein Sec61 subunit beta (Sec61ß), and coatomer protein complex subunit gamma 2 (COPG2) in porcine oocytes. The cumulus-oocyte complexes were collected from follicle with 3-6 mm in diameter. They were incubated in medium with/without EGF for 22 h (IVMⅠ) and subsequently incubated hormone-free medium with/without EGF for 22 h (IVMⅡ). Nuclear maturation state was checked by aceto-orcein stain. Protein expression of EGFR, GM-130, Sec61ß, and COPG2 were measured by immunofluorescence. In results, nuclear maturation of oocytes in EGF non-treated oocytes were significantly lower than EGF-treated groups at IVMⅠ or IVMⅡ stage (P<0.05), whereas maturational rate in EGF treatment groups at both of IVM stage was higher in among the all treatment groups (P<0.05). EGFR, GM-130, Sec61ß and COPG2 were expressed in the cytoplasm of oocytes. Especially, GM-130 and EGFR were strongly expressed, but Sec61ß and COPG2 were weakly expressed in cortical area of cytoplasm. The protein level of GM-130, Sec61ß, and COPG2 were significantly higher in the EGF-treated groups (P<0.05). However EGFR was no difference between non EGF-treated groups and control. In conclusion, EGF plays an important role in the systems for oocyte maturation with endoplasmic reticulum and Golgi apparatus. In addition, the protein levels of Sec61ß and COPG2 could be changed by EGF in the porcine oocytes during maturation.

Effect of Alpha-Linolenic Acid on Oocyte Maturation and Embryo Development in Pigs.

The aim of this study was to determine the effect of additional alpha-linolenic acid (ALA) supplementation during in vitro maturation (IVM) and culture (IVC) on nucleic maturation and embryo development of pigs. Cumulus-oocyte complexes (COCs) were incubated in IVM medium containing different concentration of ALA (25, 50 and 100 µM) for 44 h. After in vitro maturation, nuclear maturation of oocytes were evaluated by aceto-orcein stain. Mature oocytes with 50 µM ALA were fertilized and cultured in IVC medium with ALA (25, 50 and 100 µM) during early-embryogenesis (48 hours after fertilization). Then, embryos were cultured with 25 µM ALA during early embryogenesis and/or late embryogenesis (120 hours after early-embryogenesis). In results, oocyte maturation were significantly increased by 50 µM ALA treatment groups compared with control groups (p<0.05). Treatment of 25 µM ALA during early-embryogenesis enhanced cleavage rate of embryo compared with other groups (p<0.05), whereas formation and total cell number of blastocyst had no significant difference. Similarly, cleavage rate of embryos were increased by 25 µM ALA supplement during early- or late-embryogenesis than ALA treatment both stage of embryogenesis (p<0.05), but did not influence to blastocyst formation. Interestingly, total cell number of blastocyst were enhanced in ALA treatment group during early-embryogenesis. These findings indicated that ALA supplement enhance the nuclear maturation of oocyte and embryo development, however, excessive ALA could negatively influence. Therefore, we suggest that ALA is used for improvement of in vitro production of mammalian embryo and further study regarding with functional mechanism of ALA is needed.

Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment.

Generally, self-renewal of spermatogonial stem cells (SSCs) is maintained in vivo in a three-dimensional (3D) microenvironment consisting of the seminiferous tubule basement membrane, indicating the importance of the 3D microenvironment for in vitro culture of SSCs. Here, we report a 3D culture microenvironment that effectively maintains porcine SSC self-renewal during culture. Porcine SSCs were cultured in an agarose-based 3D hydrogel and in 2D culture plates either with or without feeder cells. Subsequently, the effects of 3D culture on the maintenance of undifferentiated SSCs were identified by analyzing cell colony formation and morphology, AP activity, and transcriptional and translational regulation of self-renewal-related genes and the effects on proliferation by analyzing cell viability and single cell-derived colony number. The 3D culture microenvironment constructed using a 0.2% (w/v) agarose-based 3D hydrogel showed the strongest maintenance of porcine SSC self-renewal and induced significant improvements in proliferation compared with 2D culture microenvironments. These results demonstrate that self-renewal of porcine SSCs can be maintained more effectively in a 3D than in a 2D culture microenvironment. Moreover, this will play a significant role in developing novel culture systems for SSCs derived from diverse species in the future, which will contribute to SSC-related research.

Comparison of the characteristics and multipotential and in vivo cartilage formation capabilities between porcine adipose-derived stem cells and porcine skin-derived stem cell-like cells.

OBJECTIVE: To compare the characteristics and multipotential and in vivo cartilage formation capabilities of porcine adipose-derived stem cells (pASCs) with those of porcine skin-derived stem cell-like cells (pSSCs). ANIMALS: Three 6-month-old female pigs and four 6-week-old female athymic mice. PROCEDURES: Adipose and skin tissue specimens were obtained from each pig following slaughter and digested to obtain pASCs and pSSCs. For each cell type, flow cytometry and reverse transcription PCR assays were performed to characterize the expression of cell surface and mesenchymal stem cell markers, and in vitro cell cultures were performed to determine the adipogenic, osteogenic, and chondrogenic capabilities. Each cell type was then implanted into athymic mice to determine the extent of in vivo cartilage formation after 6 weeks. RESULTS: The cell surface and mesenchymal stem cell marker expression patterns, multipotential capability, and extent of in vivo cartilage formation did not differ significantly between pASCs and pSSCs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that pSSCs may be a viable alternative to pASCs as a source of progenitor cells for tissue engineering in regenerative medicine.