Hyuk Song

Establishment and in vitro culture of porcine spermatogonial germ cells in low temperature culture conditions

The objective of this study was to establish a porcine spermatogonial germ cell (pSGC) line and develop an in vitro culture system. Isolated total testicular cells (TTCs) from 5-day-old porcine testes were primary cultured at 31, 34, and 37°C. Although the time of colony appearance was delayed at 31°C, strong alkaline phosphatase staining, expressions of pluripotency marker genes such as OCT4, NANOG, and THY1, and the gene expressions of the undifferentiated germ cell markers PLZF and protein gene product 9.5 (PGP9.5) were identified compared to 34 and 37°C. Cell cycle analysis for both pSGC and feeder cells at the three temperatures revealed that more pSGCs were in the G2/M phase at 31°C than 37°C at the subculture stage. In vitro, pSGCs could stably maintain undifferentiated germ cell and stem cell characteristics for over 60days during culture at 31°C. Xenotransplantation of pSGCs to immune deficient mice demonstrated a successful colonization and localization on the seminiferous tubule basement membrane in the recipient testes. In conclusion, pSGCs from neonatal porcine were successfully established and cultured for long periods under a low temperature culture environment in vitro.

p38 MAPK Participates in Muscle-Specific RING Finger 1-Mediated Atrophy in Cast-Immobilized Rat Gastrocnemius Muscle

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

Characterization of GFRα-1-Positive and GFRα-1-Negative Spermatogonia in Neonatal Pig Testis

Type A spermatogonia, including spermatogonial stem cells, are primary cells that maintain spermatogenesis and produce spermatozoa. Many spermatogonial markers have been reported in rodents. However, few markers have been identified in pig spermatogonia. Despite the lack of information, it is necessary to separate pure spermatogonial cells from whole testicular cells to understand the mechanism of spermatogenic meiosis and to establish spermatogonial stem cells for further biotechnological studies. The purpose of this study was to identify glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1) as a surface marker for early spermatogonia in neonatal pig testes. Histological analysis of 3-day-old pig testes revealed that type A spermatogonia, which lack heterochromatin, could be distinguished in neonatal pig testes. Immunohistochemistry of neonatal pig testes with GFRα-1 antibody identified that some of the spermatogonial cells expressed GFRα-1 on the cell membrane. Co-immunostaining with both GFRα-1 and protein gene product 9.5 (PGP 9.5) detected PGP 9.5 in all spermatogonia of neonatal pig testes, whereas GFRα-1 was not detected on the surface of some PGP 9.5-positive cells, indicating that some of the spermatogonial cells were PGP 9.5 positive and GFRα-1 negative. After immunomagnetic cell sorting using a GFRα-1 antibody, both GFRα-1-positive and GFRα-1-negative cells expressed PGP 9.5. Identifying the differential mRNA expression of both GFRα-1-positive and GFRα-1-negative cells using reverse transcription-polymerase chain reaction analysis revealed the expression of promyelocytic leukaemia zinc finger, octamer-binding protein 4 and homeobox transcription factor in both cell types. These results suggest that GFRα-1-positive and GFRα-1-negative spermatogonia exist in PGP 9.5-positive spermatogonia during the early stage of pig testes spermatogenesis, and that GFRα-1 can be used for sorting PGP 9.5-expressing spermatogonia.

Identification of multiple nuclear localization signals in murine Elf3, an ETS transcription factor

We investigated nuclear localization signal (NLS) determinants within the AT‐hook and ETS DNA‐binding domains of murine Elf3 (mElf3), a member of the subfamily of epithelium‐specific ETS transcription factors. Deletion mutants containing the AT‐hook, ETS domain or both localized strictly in the nucleus, suggesting that these individual domains contain independent NLS motif(s). Within the AT‐hook domain, four basic residues (244KRKR247) were critical for strong NLS activity, and two potent bipartite NLS motifs (236–252 and 249–267) were sufficient for nuclear import of mElf3, although less efficient than the full domain. In addition, one stretch of basic residues (318KKK320) within the ETS domain appears to be essential for mElf3 nuclear localization. Taken together, mElf3 contains multiple NLS motifs, which may function cooperatively to effect efficient nuclear transport.

Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

Protein tyrosine phosphatase SHP-2 is positively involved in platelet-derived growth factor-signaling in vascular neointima formation via the reactive oxygen species-related pathway.

The roles of Src homology domain 2-containing protein tyrosine phosphatase 2 (SHP-2) and its signaling in atherosclerosis have not been explored. Therefore, we investigated the roles of SHP-2 in the movement of rat aortic smooth muscle cells (RASMCs) and in the neointima formation of the carotid artery. Platelet-derived growth factor (PDGF)-BB (1 - 20 ng/ml) increased the activity and phosphorylation of SHP-2 and migration in RASMCs and these were suppressed by SHP-2 inhibitor NSC-87877 (30 μM) and small interfering RNA of SHP-2. PDGF-BB increased the phosphorylations of spleen tyrosine kinase (Syk) and p38 mitogen-activated protein kinase (MAPK), which were recovered by inhibition of SHP-2. Moreover, PDGF-BB increased the levels of reactive oxygen species (ROS) and ROS inhibitors decreased PDGF-BB-increased migration. Treatment of RASMCs with H2O2 (100 μM) increased cell migration and SHP-2 phosphorylation and also enhanced the phosphorylation levels of Syk and p38 MAPK. Oral administration of NSC-87877 (10 mg/kg) significantly suppressed neointima formation in a rat model of carotid artery injury. These results suggest that the activity of SHP-2 is controlled by ROS and is positively involved in the regulation of PDGF-BB-induced RASMC migration and neointima formation.

Soluble form of vascular cell adhesion molecule 1 induces migration and proliferation of vascular smooth muscle cells.

Background: Serum levels of soluble vascular cell adhesion molecule 1 (sVCAM-1) shed from its membrane-bound form are elevated in hypertension. This study clarified the effects of sVCAM-1 on vascular responses in rat aortic smooth muscle cells (RASMCs). Methods: Boyden chamber, 5-bromo-2′-deoxyuridine incorporation and ex vivo aortic ring assays for migration and proliferation, and Western blot for the kinase activity were used. Results: Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were compared functionally. sVCAM-1 increased RASMC migration and proliferation, which were greater in SHR compared with WKY rats. RASMCs expressed the very late antigen 4α receptor integrin with no difference between SHR and WKY rats. Inhibitors of phosphoinositide kinase 3 (PI3K) and spleen tyrosine kinase (Syk) and small interference RNA-Syk abolished the sVCAM-1-induced migration, proliferation and phosphorylation of focal adhesion kinase. The phosphorylation of Syk was significantly greater in RASMCs from SHR than from WKY rats. sVCAM-1 increased aortic sprout outgrowth, which was inhibited by inhibitors of PI3K and Syk. Conclusions: This study suggests that sVCAM-1 promotes the RASMC migration and proliferation via the focal adhesion kinase pathway regulated by Syk and PI3K, and the altered sVCAM-1-induced responses during hypertension are closely associated with the increments in intracellular signal transmission.

Regulation of human growth and differentiation factor 3 gene expression by NANOG in human embryonic carcinoma NCCIT cells

We investigated transactivation by NANOG in regulating growth and differentiation factor 3 (GDF3) expression in NCCIT cells. GDF3 expression was affected by shRNA‐mediated downregulation and by exogenous overexpression of NANOG specifically, as well as by retinoic acid‐mediated differentiation. GDF3 transcription was activated by NANOG, and the upstream region (−183 to −1) was sufficient to induce minimal transcriptional activity. Moreover, NANOG binds to the GDF3 minimal promoter in vivo and the transcriptional activity is mediated by NANOG transactivation domain. This study provides the first evidence that NANOG is a transcriptional activator of the expression of the oncogenic growth factor GDF3 in embryonic carcinoma cells.

Intraovarian transplantation of primordial follicles fails to rescue chemotherapy injured ovaries

Busulfan and cyclophosphamide (B/C)-treated mice exhibited a marked increase in apoptosis and a concomitant decrease in the ovarian weight. Histological and RT-PCR analysis indicate that the period of germ cell depletion in the B/C-treated ovaries coincides with decreased expression of genes Figla, Lhx8, Nobox, c-kit, and Sox3. However, depletion of the ovarian germ cells is mediated by autophagy-independent pathways that involve Fas/FasL-, TNF-, and/or p53-signalings. Treatment with B/C resulted in the cease of the reproductive function to produce their offspring during the 15th week post-treatment period in female mice. Furthermore, injection of the 3 × 106 GFP positive primordial follicles into the ovaries of the B/C treated mouse did not show apparent colonization of the transplanted follicles within the recipient ovaries. The present results suggest that B/C treatment is closely associated with an increased risk of premature ovarian failure.

Identification of metaphase II-specific gene transcripts in porcine oocytes and their expression in early stage embryos.

Annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) was used to identify the genes that are specifically or prominently expressed in porcine oocytes at the metaphase II (MII) and germinal vesicle (GV) stages. By using 60 ACPs, 13 differentially expressed genes (DEGs) were identified. The cloned genes or expressed sequence tags (ESTs) showed sequence similarity with known genes or ESTs of other species in GenBank. The mRNA expression during oocyte maturation and early embryonic development in both pigs and mice of four of these genes (namely transcription factor TZP, annexin A2, hypoxia-inducible protein 2, and ATPase 6) was further characterised by real-time quantitative reverse transcription-PCR. All four genes were markedly upregulated in pig and mouse MII oocytes compared with GV-stage oocytes. The expression levels of the four genes decreased gradually during early cleavage. Thus, these genes may play important roles during oocyte maturation and/or early cleavage in mammals. Although the detailed functions of these genes remain to be determined, their identification in the present study provides insights into meiotic maturation and fertilisation.